non-target-site based tolerance to herbicides in

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Theses and Dissertations

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Non-Target-Site Based Tolerance to Herbicides inAmaranthus palmeriReiofeli Algodon Salas-PerezUniversity of Arkansas, Fayetteville

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Non-Target-Site Based Tolerance to Herbicides in Amaranthus palmeri

A dissertation submitted in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy in Crop, Soil, and Environmental Sciences

by

Reiofeli A. Salas-Perez

Leyte State University

Bachelor of Science in Agricultural Chemistry, 2004

University of Arkansas

Master of Science in Crop, Soil, and Environmental Sciences, 2012

May 2018

University of Arkansas

This dissertation is approved for recommendation to the Graduate Council

_______________________________

Nilda R. Burgos, Ph.D.

Dissertation Director

_______________________________ _______________________________

Robert Scott, Ph.D. Nathan Slaton, Ph.D.

Committee member Committee member

_______________________________ _______________________________

Andronikos Mauromoustakos, Ph.D. Amy Lawton-Rauh, Ph.D.

Committee member Committee member

_______________________________

Franck Dayan, Ph.D.

Ex-officio member

ABSTRACT

Palmer amaranth, one of the most aggressive and damaging broadleaf weeds in the USA,

has evolved resistance to multiple herbicide modes of action. The overall objective of this

research was to elucidate the mechanisms by which Palmer amaranth adapt to herbicide selection

stress. This research aimed (1) to evaluate the efficacy of fomesafen, glufosinate, glyphosate and

trifloxysulfuron to Amaranthus populations; (2) identify candidate genes for endowing tolerance

to glufosinate; (3) investigate the involvement of non-target-site resistance (NTSR) mechanism

in an ALS-resistant population; and (4) to examine the molecular basis of resistance to PPO

inhibitors in Palmer amaranth populations from Arkansas. For objective 1, a total of 124

populations were collected in Arkansas between 2008 and 2015. Overall, 33%, 81%, and 100%

of the populations were resistant to fomesafen, glyphosate, and trifloxysulfuron, respectively.

Thirty percent of the populations were multiple resistant to fomesafen, glyphosate, and

trifloxysulfuron. All populations were controlled >88% by glufosinate. For objective 2, the

transcriptomes of glufosinate-tolerant and –sensitive biotypes were assembled using RNA-Seq.

Thirteen candidate non-target genes were highly expressed in glufosinate-tolerant biotypes,

including glutathione S-transferase (GST), two cytochrome P450, and nine additional genes

related to stress signaling and detoxification. Validation of differential gene expression by

quantitative real-time PCR revealed increased expression CYP72A219 and GST in glufosinate-

treated tolerant biotypes, indicating their involvement in glufosinate tolerance. For objective 3, a

population with cross resistance to multiple ALS-inhibiting herbicides was investigated. Two of

the nine resistant plants harbored Ser653Asn mutation in the ALS gene. Resistant plants that

lacked ALS mutations had elevated levels of CYP81B and GSTF10 genes. This Palmer amaranth

population from Arkansas exhibit both target-site (TS) and NTSR to ALS inhibitors. For

objective 4, resistance to PPO inhibitors was first detected in a population collected in 2011 with

resistance attributed to PPO Gly210 deletion. Several PPO-resistant populations were confirmed

in 2014 and 2015; the majority (55%) of the resistant biotypes carried the same mutation. An

alternative target-site mutation Arg128Gly was also identified in at least one population. Overall,

this research showed that Palmer amaranth has multiple genetic adaptation traits to counteract

the lethal effects of herbicides.

ACKNOWLEDGEMENTS

I am incredibly grateful to my Lord and Saviour Jesus Christ for his provision and unfailing

mercies. I would not have continued the journey if not of His grace and strength that sustained

me throughout my studies.

Sincere thanks to my advisor, Dr. Nilda R. Burgos, for giving me the opportunity to pursue

graduate studies under her guidance. I thank her for the constant encouragement and support she

provided in my entire PhD journey.

I also thank my graduate advisory committee members, Dr. Robert C. Scott, Dr. Nathan Slaton,

Dr. Andy Mauromoustakos, Dr. Amy Lawton-Rauh, and Dr. Franck Dayan for their patience,

support, and constructive comments that contributed towards the improvement of this

manuscript.

Special thanks to all my Weed Physiology colleagues Gulab Rangani, Seth Bernard Abugho, Jun

Estorninos, George Botha, Vijay Singh, Shilpa Singh, Ean Alcober, Hussain Tahir, Ana Claudia

Langaro, Marlon Bastiani, Matheus Noguera, Eduardo Chagas, JP Refatti, Leo Piveta, Claudia

Oliveira, Tiago Kaspary, Fernando Ramirez, Fernando Martini, Caroline Bevilacqua, Ana

Carolina Roso, Christopher Rouse, Teal Penka, Josiane Argenta, Pâmela Carvalho de Lima, and

Sirichai for extending their help in my projects. Their camaraderie made my student life

enjoyable.

I am also grateful to Duyen, Sangeeta, Dennis, Sittie, Samantha and to my KAMPI/FSO,

NWAFAA and Grace Fellowship friends for their unwavering support, the fun-filled memories

and the prayers.

I also thank the various staffs and fellow graduate students of the CSES department for the

assistance and friendly conversations. Finally, I would like to thank Cotton Incorporated and the

Arkansas Soybean Promotion Board for funding my research, as well as BASF for the internship

opportunity.

DEDICATION

This work is dedicated to my husband (Roderic Perez) and my parents (Felix and Rosario Salas)

for their love, prayers, and support throughout the degree program. Also, I thank and pay

gratitude to my siblings and in-laws for their love, support, and trust which kept me moving.

Especially, I would like this opportunity to thank my beloved husband, the wind beneath my

wings, for being there through thick and thin, for never giving up on me, and for constantly

encouraging and motivating me to finish the race triumphantly.

TABLE OF CONTENTS

CHAPTER I .................................................................................................................................. 1

INTRODUCTION .............................................................................................................. 1

Literature Cited ................................................................................................................... 8

CHAPTER II ............................................................................................................................... 11

REVIEW OF LITERATURE ........................................................................................... 11

Palmer amaranth ................................................................................................... 12

Non-target-site based resistance mechanims ........................................................ 14

Glufosinate ............................................................................................................ 17

ALS-inhibiting herbicides ..................................................................................... 19

PPO-inhibiting herbicides ..................................................................................... 22

Literature Cited ................................................................................................................. 24

CHAPTER III ............................................................................................................................. 31

RNA-SEQ TRANSCRIPTOME ANALYSIS OF AMARANTHUS PALMERI WITH

DIFFERENTIAL TOLERANCE TO GLUFOSINATE HERBICIDE ................................ 31

Abstract ............................................................................................................................. 32

Introduction ....................................................................................................................... 34

Materials and Methods ...................................................................................................... 36

Results ............................................................................................................................... 46

Discussion ......................................................................................................................... 53

Literature Cited ................................................................................................................. 64

CHAPTER IV.............................................................................................................................. 95

MULTIPLE RESISTANCE TO ALS, PPO, AND EPSPS INHIBITORS IN

AMARANTHUS PALMERI FROM ARKANSAS, USA.......................................................... 95

ABSTRACT ...................................................................................................................... 96

INTRODUCTION ............................................................................................................ 97

MATERIALS AND METHODs .................................................................................... 100

RESULTS AND DISCUSSION ..................................................................................... 106

REFERENCES ............................................................................................................... 114

CHAPTER V ............................................................................................................................. 128

RESISTANCE TO PPO-INHIBITING HERBICDE IN PALMER AMARANTH FROM

ARKANSAS............................................................................................................................... 128

Abstract ....................................................................................................................................... 129

INTRODUCTION .......................................................................................................... 130

MATERIALS AND METHODS .................................................................................... 132

RESULTS AND DISCUSSION ..................................................................................... 137

REFERENCES ............................................................................................................... 144

CHAPTER VI............................................................................................................................ 152

FREQUENCY OF GLY210 DELETION MUTATION AMONG

PROTOPORHYRINOGEN OXIDASE INHIBITOR-RESISTANT PALMER

AMARANTH (AMARANTHUS PALMERI) POPULATIONS ............................................ 152

Abstract ........................................................................................................................... 153

Introduction ..................................................................................................................... 155

Materials and Methods .................................................................................................... 157

Results and Discussions .................................................................................................. 162

Literature Cited ............................................................................................................... 172

CONCLUSIONS ....................................................................................................................... 189

LIST OF TABLES

CHAPTER III

Table 1. Cluster analysis of A. palmeri accessions treated with glufosinate at 0.55 kg ha-1. ...... 74

Table 2. Response of A. palmeri (08-Lee-C) to foliar-applied herbicides. ................................. 75

Table 3. Glufosinate dose required to reduce growth by 50% (GR50) in A. palmeri 08-Lee-

C, C1 and SS accessions. ............................................................................................ 76

Table 4. Summary of statistics for transcriptome assembly. ....................................................... 77

Table 5. Differentially expressed genes putatively involved in differential tolerance to

glufosinate in Amaranthus palmeri. ............................................................................ 78

Table 6. Upregulated genes in glufosinate-treated and non-treated tolerant (T) plants, and in

glufosinate-treated T relative to treated susceptible (S) plants, assigned with

Gene Ontology molecular function and biological process related to metabolism

and signaling pathways. .............................................................................................. 79

Table 7. Candidate non-target genes, identified by RNA-Seq analysis, that are potentially

involved in conferring differential tolerance to glufosinate in A. palmeri. ................ 82

CHAPTER IV

Table 1. Primers used in the gene expression analysis by qRT-PCR. ........ Error! Bookmark not

defined.

Table 2. Response of 15Cri-B population to the normal dose of ALS (flumetsulam,

imazethapyr, primisulfuron, pyrithiobac, trifloxysulfuron), fomesafen, glyphosate

and glufosinate herbicides.. .......................................... Error! Bookmark not defined.

Table 3. Level of resistance to ALS, PPO, and EPSPS inhibitors in 15Cri-B Palmer

amaranth population..................................................... Error! Bookmark not defined.

Table 4. Effect of pretreatment of P450 inhibitor on the phytotoxicity of trifloxysulfuron (8

g ha-1)in 15Cri-B Palmer amaranth population. ........... Error! Bookmark not defined.

Table 5. Cytochrome P450 genes involved in NTSR to ALS inhibitors in various weed and

crop species………………………………………………………………………….125

CHAPTER V

Table 1. GR50 and LD50 values of original, C1, and C2 AR11-LAW-B populations in

Arkansas, USA. ......................................................................................................... 147

Table 2. Response of Amaranthus palmeri AR11-LAW-B population to foliar-applied

herbicides other than protoporphyrinogen oxidase inhibitors, Arkansas, USA. ....... 148

Table 3. GR50 values and resistance levels to ALS inhibitors in AR11-LAW-B Palmer

amaranth population in Arkansas, USA.................................................................... 149

CHAPTER VI

Table 1. Number of Palmer amaranth accessions in Arkansas, USA tested with foliar-

applied fomesafen at 263 g ha-1. ............................................................................... 175

Table 2. Common, trade names, and manufacturers of herbicides used in the study. .............. 176

Table 3. Response of fomesafen-resistant Palmer amaranth accessions to the recommended

rate of various foliar-applied PPO herbicides. .......................................................... 178

Table 4. Herbicide resistance profile of PPO-resistant Palmer amaranth accessions to other

foliar-applied non-PPO herbicides. ........................................................................... 180

Table 5. GR50 values of PPO-resistant Palmer amaranth accessions in Arkansas. ................... 181

LIST OF FIGURES

CHAPTER III

Figure 1. Shoot biomass reduction (%) of 08-Lee-C, C1, and SS A. palmeri accessions, 21

days after glufosinate treatment. Data were best described with nonlinear, sigmoidal, four-

parameter logistic regression function. ......................................................................................... 83

Figure 2. Ammonia content in glufosinate-tolerant (T) and –susceptible (S) A. palmeri.Error

bars represent standard error. White bars = S plants; gray bars = T plants. ................................. 84

Figure 3. Relative copy number of A. palmeri GS2 in glufosinate-susceptible (S) and -

tolerant (T) plants. Error bars represent standard deviation of the mean. Gray bars = T

plants; black bars = T plant. .......................................................................................................... 85

Figure 4. Multiple alignment of the plastidic glutamine synthetase (GS2) amino acid

sequences in Amaranthus. A. palmeri (reference A. palmeri), T1 and T2 = GS2 alleles of

glufosinate-tolerant A. palmeri biotype, S = GS2 sequence of glufosinate-susceptible A.

palmeri biotype. ............................................................................................................................ 86

Figure 5. Multidimensional scaling (MDS) plot showing the relationship between sample

types.TWO = non-treated tolerant, TWT= treated tolerant, SWO = non-treated susceptible,

SWT = treated susceptible. ........................................................................................................... 87

Figure 6. Volcano plots depicting differential gene expression between treatments. A)

Treated susceptible (S) relative to non-treated S plants (SWT vs SWO), B) Treated tolerant

(T) relative to treated S plants (TWT vs SWT), C) treated T relative to non-treated T plants

(TWT vs TWO), and D) treated T plants relative to non-treated S plants (TWO vs SWO).

The x-axis shows the log fold change or relative abundance. The P value (-log base 10) for

differential gene expression is plotted on the y axis. Black dots represent genes that did not

change in expression; colored dots on the left indicate genes with significantly

downregulated expression; colored dots on the right indicate genes with significantly

upregulated expression.................................................................................................................. 88

Figure 7. The number of differentially expressed genes common or specific to treated and

non-treated T and S plants.A 4-way Venn diagram depicting the distribution of differentially

expressed genes across all pairwise comparisons. The number within each shaded area is the

number of differentially expressed genes common between treatments. ..................................... 89

Figure 8. Biological processes (A) and molecular functions (B) of upregulated genes in

treated T relative to treated S plants. ............................................................................................ 90

Figure 9. Biological processes (A) and molecular functions (B) of downregulated genes in

treated T relative to treated S plants. ............................................................................................ 91

Figure 10. Biological processes (A) and molecular functions (B) of differentially expressed

genes that are common in treated T relative to treated S and non-treated T plants. ..................... 92

Figure 11. Heat map analysis of genes that are putatively related to abiotic stress response in

A. palmeri.TWO (non-treated tolerant), SWO (non-treated susceptible), TWT (glufosinate-

treated tolerant), SWT (glufosinate-treated susceptible). ............................................................. 93

Figure 12. Gene expression fold-change of seven candidate NTS genes in glufosinate-

tolerant Amaranthus palmeri relative to –susceptible ones. Tolerant and susceptible plants

from two populations were identified based on the level of injury incurred after glufosinate

treatment.GST and CYP72A219 was highly expressed in the tolerant biotypes in at least one

population. Untreated = before glufosinate treatment, Treated = 24 h after glufosinate

treatment. Error bars represent standard error from six biological replicates. HSP = heat

shock protein, GST = glutathione S-transferase, NMO = nitronate monooxygenase, ETF =

ethylene-responsive transcription factor, ABC = ABC transporter, NAC = NAC

transcription factor, CYP72A219 = cytochrome P450 CYP72A219. .......................................... 94

CHAPTER IV

Figure 1. Amino acid sequence alignment of acetolactate synthase (ALS) in sensitive (SS)

and resistant (15Cri-B) Palmer amaranth plant samples.Two resistant plants harbored

Ser653Asn (AGC to AAC) mutation. .............................................. Error! Bookmark not defined.

Figure 2. Gene expression analysis of ALS and NTSR genes in trifloxysulfuron-resistant

(15Cri-B) and –susceptible (SS) Palmer amaranth biotypes. Fold-change (2-ΔCt) in gene

expression was calculated relative to the susceptible samples, where ΔCt = [Ct target-gene -

Ct mean of internal control genes]. Means and standard errors from three biological

replicates are shown.. ..................................................................... Error! Bookmark not defined.

CHAPTER V

Figure 1. Shoot biomass reduction (%) of PPO-resistant and –susceptible Palmer amaranth

population, 21 d after fomesafen treatment.Treatment means (n = 10) + 1 standard error are

plotted and fitted to a regression curve. Data were best described with a nonlinear,

sigmoidal, three-parameter Gompertz regression function, y =a*exp{-exp[-b*(x-c0]}. ........... 150

Figure 2. Dose-response curves of PPO-resistant and –susceptible Palmer amaranth

population from Arkansas. Mortality (%) were obtained at 21 d after herbicide treatment.

Treatment means (n = 10) + 1 standard error are plotted and fitted to a regression curve.

Data were best described with a nonlinear, sigmoidal, three-parameter Gompertz regression

function, y =a*exp{-exp[-b*(x-c0]}. .......................................................................................... 151

CHAPTER VI

Figure 1. Variability in response to foliar-applied fomesafen (263 g ha-1) among Palmer

amaranth accessions collected between 2008 and 2015. Box plot shows median values

(horizontal line inside the box), mean values (marked X), first and third quartile values (box-

outlines), minimum and maximum values (whiskers), and outlier values (open circles). .......... 182

Figure 2. Frequency of fomesafen-resistant plants in Palmer amaranth accessions collected

in 2015 from Arkansas. Fomesafen at 263 g ha-1 was applied with 0.5% non-ionic surfactant

to 7.6-cm tall seedlings. Survivors are categorized based on visible injury. .............................. 183

Figure 3. Hierarchal clustering of 2014 and 2015 Palmer amaranth accessions treated with

foliar-applied fomesafen at 263 g ha-1.The herbicide was applied with 0.5% non-ionic

surfactant to 7.6-cm tall seedlings. Cluster 1 = most sensitive to fomesafen (50 accessions),

Cluster 2 = resistant to fomesafen with majority of survivors incurred >60% injury (19

accessions), Cluster 3 = resistant to fomesafen, majority of survivors sustained 11-30%

injury (2 accessions), Cluster 4 = resistant to fomesafen, majority of survivors incurred 0-

10% injury (3 accessions). .......................................................................................................... 184

Figure 4. (A) Cluster analysis of mortality levels in Palmer amaranth accessions collected in

2014 and 2015 treated with 870 g ha-1 glyphosate. Cluster 1 (n = 13 accessions; resistant to

glyphosate with majority of survivors incurred 31-60% injury), cluster 2 (n = 23 accessions;

resistant to glyphosate with majority of survivors incurred <11% injury), cluster 3 (n = 23

accessions; least sensitive to glyphosate), and cluster 4 (n = 14 accessions; resistant to

glyphosate with majority of its survivors incurred 61-89% injury) are depicted in box-plot.

Glyphosate was applied to 7.6-cm tall seedlings. (B) Variability in response to dicamba (280

g ae ha-1), glufosinate (549 g ha-1) and trifloxysulfuron (7.84 g ha-1) among Palmer amaranth

accessions collected in 2014 and 2015 from Arkansas, USA. Box plot shows median values

(horizontal line inside the box), mean values (marked with X), first and third quartile values

(box-outlines), minimum and maximum values (whiskers), and outlier values (open circles). . 185

Figure 5. Herbicide resistance profiles of Palmer amaranth populations from Arkansas

sampled in 2014 and 2015. ......................................................................................................... 186

Figure 6. Counties with confirmed PPO-resistant Palmer amaranth in Arkansas.Counties that

are shaded had at least one field-population with PPO-resistant Palmer amaranth biotypes

carrying the Gly210 deletion in the PPO gene.............................................................................. 187

Figure 7. Prevalence of PPO Gly210 mutation in PPO-resistant Palmer amaranth from

Arkansas. (A) Pie-chart displaying the percentage of PPO-resistant accessions that contained

and lacked the PPO Gly210 mutation. Black = all fomesafen survivors in these accessions

contained the Gly210 mutation. White = all survivors in these accessions lacked the Gly210

mutation. Gray = survivors in these accessions contained mixture of carriers and non-

carriers of Gly210 mutation. (B) Proportion of Gly210 mutation carriers in each PPO-resistant

accession. .................................................................................................................................... 188

LIST OF PUBLISHED ARTICLES

CHAPTER III

Reiofeli A. Salas, Christopher A. Saski, Rooksana E. Noorai, Subodh K. Srivastava, Amy

Lawton-Rauh, Robert L. Nichols, and Nilda R. Burgos. 2018. RNA-Seq transcriptome analysis

of Amaranthus palmeri with differential tolerance to glufosinate herbicide. PLoS ONE

(submitted)----------------------------------------------------------------------------------------------------31

CHAPTER V

Reiofeli A. Salas, Nilda R. Burgos, Patrick J. Tranel, Shilpa Singh, Les Glasgow, Robert C. Scott

and Robert L. Nichols. 2016. Resistance to PPO-inhibiting herbicide in Palmer amaranth from

Arkansas. Pest Management Science 72: 864-869. DOI: 10.1002/ps.4241------------------------128

CHAPTER V1

Reiofeli A. Salas-Perez, Nilda R. Burgos, Gulab Rangani, Shilpa Singh, Joao Paulo Refatti,

Leonard Piveta, Patrick J. Tranel, Andy Mauromoustakos, and Robert C. Scott. 2017. Frequency

of Gly-210 deletion mutation among protoporphyrinogen oxidase inhibitor-resistant Palmer

amaranth (Amaranthus palmeri) populations. Weed Science 65: 718-731. DOI:

10.1017/wsc.2017------------------------------------------------------------------------------------------152

1

CHAPTER I

INTRODUCTION

2

Introduction

Palmer amaranth (Amaranthus palmeri S Watson) is one of the most common,

troublesome, and economically damaging agronomic weeds throughout the southern US (Ward

et al., 2013). The competitive ability of Palmer amaranth is attributed to its fast growth rate (Jha

et al., 2008), high fecundity (Keeley et al., 1987), good light interception, and high water use

efficiency (Ehleringer, 1983). With estimates of over 600,000 seeds per plant, it can replenish the

seedbank tremendously (Keeley et al., 1987). Because it competes effectively with crops for

nutrients, water, light and space, it can significantly reduce crop yield. Palmer amaranth densities

of 8 and 9 plants/m2 can reduce soybean (Glycine max) yield by 78% (Bensch et al., 2003) and

corn (Zea mays) grain yield by 91% (Massinga et al., 2001), respectively. Palmer amaranth

interference for 63 d after crop emergence can cause 77% yield loss in cotton (Gossypium

hirsutum) (Fast et al., 2009). Apart from reducing yield, heavy infestation of Palmer amaranth

interfere with crop harvest and increased harvesting time by two- to four-fold (Smith et al.,

2000).

Palmer amaranth control has become a challenge because of limited herbicide resources

and its remarkable tendency to evolve herbicide resistance. As a dioecious species, Palmer

amaranth is outcrossing, allowing herbicide resistance to spread rapidly (Steckel, 2007).

Glyphosate resistance trait can be transferred up to 300 m through pollen from glyphosate-

resistant males to glyphosate-susceptible female plants (Sosnoskie et al., 2012). To date, Palmer

amaranth has been confirmed resistant to six different modes of action: acetolactate synthase

(ALS) inhibitors, dinitroanilines, triazine, glyphosate, hydroxyphenylpyruvate dioxygenase

(HPPD) inhibitors, and most recently to protoporphyrinogen oxidase (PPO) inhibitors (Heap

2018). This narrows down available control option for resistant populations. PPO herbicides like

3

Valor and Reflex are intensively used to combat herbicide-resistant Palmer amaranth. Losing

these herbicides to resistance problems would severely hamper efforts to manage weeds.

Upcoming herbicide-resistant (HR) crop technology with resistance to glyphosate and dicamba,

to glufosinate, to bleacher herbicides and other trait combinations are being developed. However,

several weed species already reported resistance to some of these herbicides (Heap 2018).

Knowing the response of Palmer amaranth populations to alternative herbicides in HR crops will

refine herbicide recommendations for resistance management.

Herbicide resistance is the inherited ability of the plant to survive and reproduce

following exposure to a dose of herbicide that would normally be lethal to the wild type (WSSA,

1998). Resistance is essentially a natural phenomenon which occurs spontaneously in weed

populations, but is only noticed when a selection pressure is applied to the weeds through the

application of a herbicide. Resistance to herbicides is the result of weed evolutionary adaptation

to herbicide application (Delye, 2013). There are two primary mechanisms of herbicide

resistance in weeds: target-site and non-target-site resistance. Target-site resistance (TSR) is

caused by alteration of the gene encoding the target protein of the herbicide. Non-target-site

resistance (NTSR) is due to mechanism(s) that minimize the amount of herbicide reaching the

target site (Powles and Yu, 2010).This includes decreased herbicide penetration and

translocation, increased herbicide sequestration, and metabolism. A study reported that rigid

ryegrass resistance to nine modes of action is attributed to NTSR (Burnet et al., 1994). Previous

research has indicated that low-dose herbicide application tend to promote non-target-site

metabolic resistance (Gardner et al., 1998). Compared with target-site resistance, non-target-site

herbicide resistance poses a greater threat because it can confer unpredictable resistance to

herbicides with various modes of action and the multi-gene involvement in the mechanism (Petit

4

et al., 2010). Despite the importance of NTSR in understanding herbicide resistance evolution,

the genetic determinants of NTSR are poorly understood because of the inherently complicated

biochemical processes and the limited genomic information available in weedy species (Delye,

2013; Yuan et al., 2007). However, the recent advances in genomics technologies will enable

identification of specific genes that mediate NTSR mechanism.

Acetolactate synthase inhibitors have been widely used for Palmer amaranth control since

their introduction in 1982 (Gaeddert et al., 1997). Inhibition of ALS enzyme affects the synthesis

of branched-chain amino acids (valine, leucine, and isoleucine), ultimately leading to plant death.

There are five chemical families of herbicide that inhibit ALS: sulfonylureas (SUs),

imidazolinones (IMIs), triazolopyrimidines (TPs), sulfonylaminocarbonyltriazolinones (SCTs),

pyrimidinylthiobenzoates (PTBs). The ALS herbicides are widely used because they provide

broad-spectrum weed control at low doses, soil residual activity, wide application windows,

excellent crop safety and low mammalian toxicity. However, these herbicides have high

propensity to select resistant weed populations due to its widespread usage, strong selection

pressure, and high levels of natural variability in the ALS gene (Tranel and Wright, 2002). To

date, 160 species including Palmer amaranth are resistant to ALS-inhibiting herbicides (Heap

2018). Populations of Palmer amaranth resistant to ALS inhibitors are prevalent and have been

identified in 14 states in the US, including Arkansas, Arizona, Delaware, Florida, Georgia,

Illinois, Kansas, Maryland, Michigan, Mississippi, Tennessee, North Carolina, South Carolina,

and Wisconsin (Nandula et al., 2012; Salas-Perez et al., 2017; Sosnoskie et al., 2011). Resistance

is frequently attributed to target-site mutation. However, various studies have reported the

presence of both target-site and non-target-site resistance in some populations of Palmer

5

amaranth, Spanish corn poppy (Papaver rhoeas) and flixweed (Descurainia Sophia L.)

populations (Nakka et al., 2017; Rey-Caballero et al., 2017; Yang et al., 2016).

The widespread distribution of glyphosate-resistant weeds compelled farmers to use

alternative herbicides including another non-selective herbicide, glufosinate, to control

herbicide-resistant weeds in glufosinate-tolerant crops. Glufosinate is a fast-acting

postemergence herbicide that controls weeds by inhibiting glutamine synthetase (GS) (E.C.

6.3.1.2), the enzyme that converts glutamate and ammonia to glutamine (Wild and

Manderscheid, 1984). Inhibition of GS by glufosinate leads to ammonia accumulation, inhibition

of amino acid synthesis, and indirect inhibition of photosynthesis, ultimately leading to plant

death (Tachibana et al., 1986). To date, resistance to glufosinate has been confirmed in

goosegrass (Eleusine indica) from Malaysia (Jalaludin et al., 2010; Seng et al., 2010) and Italian

ryegrass (Lolium perenne ssp. multiflorum) from Oregon (Avila-Garcia and Mallory-Smith,

2011). An amino acid mutation in the chloroplast-encoded GS gene, Asp171Asn, conferred

resistance to glufosinate in Italian ryegrass (Avila-Garcia et al., 2012). Resistance to glufosinate

in glufosinate-resistant crops is achieved using transgenic methods to insert the bar or pat

(phosphinothricin acetyltransferase) gene from a bacterium to the plant’s genome, allowing

detoxification of glufosinate by acetylation (Droge-Laser et al., 1994).

PPO-inhibiting herbicides became widely used during 1980-1990. However, after

introduction of glyphosate-resistant crops, usage of PPO-herbicides declined until 2006 when

economically damaging weeds evolved resistance to glyphosate (USDA-NASS). These current

situations in agriculture reinforce the high interest in PPO-inhibiting herbicides in recent years

(Dayan et al., 2010; Salas et al., 2016; Salas-Perez et al., 2017). The PPO enzyme catalyzes the

oxidation of protoporphyrinogen IX (protogen) to protoporphyrin IX (proto), the last common

6

intermediate for the tetrapyrole synthesis system (Becerril and Duke, 1989; Jacobs et al., 1991).

Several classes of PPO-inhibiting herbicides such as diphenyl ethers, thiadiazoles, oxadiazoles,

triazolinones, N-phenyl-phthalimides, and pyrimidinediones inhibit the PPO enzyme. Inhibition

of PPO results in the generation of singlet oxygen species that oxidize lipid and protein

membranes, leading to plant death (Sherman et al., 1991). However, a few species of weeds have

slowly evolved resistance from the repeated use of these herbicides in past few years. To date, 11

weed species have evolved resistance against PPO-herbicides including Palmer amaranth (Heap

2018; Salas et al., 2016; Salas-Perez et al., 2017;Giacomini et al., 2017). Many molecular and

physiological mechanisms underlie the PPO-herbicide resistance, but most often, the evolution

of resistance attributed to target-site mutations (Dayan et al., 2010; Edwards, 1996; Skipsey et

al., 1997; Thinglum et al., 2011; Wuerffel et al., 2015).

Palmer amaranth control is becoming more difficult due to its adaptability, high seed

production and resistance to many herbicides used for its management. With the evolution of

Palmer amaranth populations that are resistant to herbicides of different modes of action, new

approaches should be implemented to control and reduce the frequency of herbicide-resistant

weeds. Understanding the molecular mechanisms endowing herbicide resistance will contribute

to wiser use of herbicide resources and enable innovations that, together with integrated control

strategies, will help minimize and manage herbicide-resistance evolution (Powles and Yu, 2010).

An understanding of the fundamental molecular mechanisms behind herbicide resistance is

necessary to minimize and manage resistance evolution and increase crop yield. With the recent

advances in next-generation sequencing, tools are available for characterizing genome-wide gene

expression in Palmer amaranth using transcriptomics, which is helpful in gene discovery and

understanding the molecular mechanisms involved in plant response to herbicides and herbicide

7

resistance evolution. The goal of this study is to elucidate the mechanisms by which Palmer

amaranth (or other weeds) adapt to herbicide selection stress. The specific objectives of this

research are to (1) evaluate the efficacy of foliar-applied fomesafen, glufosinate, glyphosate and

trifloxysulfuron to Amaranthus accessions collected between 2008 and 2015 in Arkansas; (2)

identify candidate genes for endowing tolerance to glufosinate; (3) investigate the involvement

of non-target-site resistance mechanism in an ALS-resistant population; and (4) to examine the

molecular basis of resistance to PPO inhibitors in Palmer amaranth populations from Arkansas.

8

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perenne) populations also exhibit resistance to glufosinate. Weed Science 59: 305-309.

Avila-Garcia, W. V., Sanchez-Olguin, E., Hulting, A. G., Mallory-Smith, C. 2012. Target-site

mutation associated with glufosinate resistance in Italian ryegrass (Lolium perenne L. ssp

multiflorum). Pest Management Science 68: 1248-1254.

Becerril, J. M., Duke, S. O. 1989. Protoporphyrin IX content correlates with activity of

photobleaching herbicides. Plant Physiol 90: 1175-81.

Bensch, C. N., Horak, M. J., Peterson, D. 2003. Interference of redroot pigweed (Amaranthus

retroflexus), Palmer amaranth (A. palmeri), and common waterhemp (A. rudis) in

soybean. Weed Science 51: 37-43.

Burnet, M. W. M., Hart, Q., Holtum, J. A. M., Powles, S. B. 1994. Resistance to nine herbicide

classes in a population of rigid ryegrass (Lolium rigidum). Weed Science 42: 369-377.

Dayan, F. E. et al. 2010. Biochemical and structural consequences of a glycine deletion in the

alpha-8 helix of protoporphyrinogen oxidase. Biochimica Et Biophysica Acta-Proteins

and Proteomics 1804: 1548-1556.

Delye, C. 2013. Unravelling the genetic bases of non-target-site-based resistance (NTSR) to

herbicides: A major challenge for weed science in the forthcoming decade. Pest

Management Science 69: 176-187.

Droge-Laser, W., Siemeling, U., Puhler, A., Broer, I. 1994. The metabolites of the herbicide L-

phosphinothricin (glufosinate): Identification, stability, and mobility in transgenic,

herbicide-resistant, and untransformed plants. Plant Physiology 105: 159-166.

Edwards, R. 1996. Characterisation of glutathione transferases and glutathione peroxidases in

pea (Pisum sativum). Physiologia Plantarum 98: 594-604.

Ehleringer, J. 1983. Ecophysiology of Amaranthus palmeri, a sonoran desert summer annual.

Oecologia 57: 107-112.

Heap I. The international survey of herbicide resistant weeds. 2018.

http://www.weedscience.com. Accessed 22 January 2018.

Fast, B. J., Murdoch, S. W., Farris, R. L., Willis, J. B., Murray, D. S. 2009. Critical timing of

Palmer amaranth (Amaranthus palmeri) removal in second-generation glyphosate-

resistant cotton. The Journal of Cotton Science 13: 32-36.

Gaeddert, J. W., Peterson, D. E., Horak, M. J. 1997. Control and cross-resistance of an

acetolactate synthase inhibitor-resistant Palmer amaranth (Amaranthus palmeri) biotype.

Weed Technology 11: 132-137.

Gardner, S. N., Gressel, J., Mangel, M. 1998. A revolving dose strategy to delay the evolution of

both quantitative vs major monogene resistances to pesticides and drugs. International

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Giacomini, D. A. et al. 2017. Two new PPX2 mutations associated with resistance to PPO-

inhibiting herbicides in Amaranthus palmeri. Pest Management Science 73: 1559-1563.

http://www.weedscience.com/

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Jacobs, J. M., Jacobs, N. J., Sherman, T. D., Duke, S. O. 1991. Effect of diphenyl ether

herbicides on oxidation of protoporphyrinogen to protoporphyrin in organellar and

plasma membrane enriched fractions of barley. Plant Physiology 97: 197-203.

Jalaludin, A., Ngim, J., Bakar, B. H. J., Alias, Z. 2010. Preliminary findings of potentially

resistant goosegrass (Eleusine indica) to glufosinate-ammonium in Malaysia. Weed

Biology and Management 10: 256-260.

Jha, P., Norsworthy, J. K., Riley, M. B., Bielenberg, D. G., Bridges, W., Jr. 2008. Acclimation of

Palmer amaranth (Amaranthus palmeri) to shading. Weed Science 56: 729-734.

Keeley, P. E., Carter, C. H., Thullen, R. J. 1987. Influence of planting date on growth of Palmer

amaranth (Amaranthus palmeri). Weed Science 35: 199-204.

Massinga, R. A., Currie, R. S., Horak, M. J., Boyer, J. 2001. Interference of Palmer amaranth in

corn. Weed Science 49: 202-208.

Nakka, S., Thompson, C. R., Peterson, D. E., Jugulam, M. 2017. Target site-based and non-target

site based resistance to ALS inhibitors in Palmer amaranth (Amaranthus palmeri). Weed

Science 65: 681-689.

Nandula, V. K. et al. 2012. Multiple resistance to glyphosate and pyrithiobac in Palmer amaranth

(Amaranthus palmeri) from Mississippi and response to flumiclorac. Weed Science 60:

179-188.

Petit, C., Duhieu, B., Boucansaud, K., Delye, C. 2010. Complex genetic control of non-target-

site-based resistance to herbicides inhibiting acetyl-coenzyme A carboxylase and

acetolactate-synthase in Alopecurus myosuroides Huds. Plant Science 178: 501-509.

Powles, S. B., Yu, Q. 2010. Evolution in Action: Plants Resistant to Herbicides. Pages 317-347

in S. Merchant, W.R. Briggs, D. Ort, eds. Annual Review of Plant Biology, Vol 61.

Rey-Caballero, J., Menéndez, J., Osuna, M. D., Salas, M., Torra, J. 2017. Target-site and non-

target-site resistance mechanisms to ALS inhibiting herbicides in Papaver rhoeas.

Pesticide Biochemistry and Physiology 138: 57-65.

Salas, R. A. et al. 2016. Resistance to PPO-inhibiting herbicide in Palmer amaranth from

Arkansas. Pest Management Science 72: 864-869.

Salas-Perez, R. A. et al. 2017. Frequency of Gly-210 deletion mutation among

protoporphyrinogen oxidase inhibitor–resistant Palmer amaranth (Amaranthus palmeri)

populations. Weed Science 65: 718-731.

Seng, C. T., Van Lun, L., San, C. T., Bin Sahid, I. 2010. Initial report of glufosinate and paraquat

multiple resistance that evolved in a biotype of goosegrass (Eleusine indica) in Malaysia.

Weed Biology and Management 10: 229-233.

Sherman, T. D. et al. 1991. Physiological-basis for differential sensitivities of plant-species to

protoporphyrinogen oxidase-inhibiting herbicides. Plant Physiology 97: 280-287.

Skipsey, M., Andrews, C. J., Townson, J. K., Jepson, I., Edwards, R. 1997. Substrate and thiol

specificity of a stress-inducible glutathione transferase from soybean. FEBS Letters 409:

370-374.

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Smith, D. T., Baker, R. V., Steele, G. L. 2000. Palmer amaranth (Amaranthus palmeri) impacts

on yield, harvesting, and ginning in dryland cotton (Gossypium hirsutum). Weed

Technology 14: 122-126.

Sosnoskie, L. M., Kichler, J. M., Wallace, R. D., Culpepper, A. S. 2011. Multiple resistance in

Palmer amaranth to glyphosate and pyrithiobac confirmed in Georgia. Weed Science 59:

321-325.

Sosnoskie, L. M. et al. 2012. Pollen-mediated dispersal of glyphosate-resistance in Palmer

amaranth under field conditions. Weed Science 60: 366-373.

Steckel, L. E. 2007. The dioecious Amaranthus spp.: Here to stay. Weed Technology 21: 567-

570.

Tachibana, K., Watanabe, T., Sekizawa, Y., Takematsu, T. 1986. Action mechanism of bialaphos

2: Accumulation of ammonia in plants treated with bialaphos. Journal of Pesticide

Science 11: 33-37.

Thinglum, K. A. et al. 2011. Wide Distribution of the waterhemp (Amaranthus tuberculatus)

delta G210 PPX2 mutation, which confers resistance to PPO-inhibiting herbicides. Weed

Science 59: 22-27.

Tranel, P. J., Wright, T. R. 2002. Resistance of weeds to ALS-inhibiting herbicides: What have

we learned? Weed Science 50: 700-712.

USDA-NASS. US Department of Agriculture, National Statistics Service.www.nass.usda.gov

[accessed March 2018]

Ward, S. M., Webster, T. M., Steckel, L. E. 2013. Palmer amaranth (Amaranthus palmeri): A

review. Weed Technology 27: 12-27.

Wild, A., Manderscheid, R. 1984. The effect of phosphinothricin on the assimilation of ammonia

in plants. Zeitschrift Fur Naturforschung C-a Journal of Biosciences 39: 500-504.

WSSA. 1998. Herbicide resistance and tolerance definitions. Weed Technology 12:789.

Wuerffel, R. J. et al. 2015. Distribution of the delta G210 protoporphyrinogen oxidase mutation

in Illinois waterhemp (Amaranthus tuberculatus) and an improved molecular method for

detection. Weed Science 63: 839-845.

Yang, Q. et al. 2016. Target-site and non-target-site based resistance to the herbicide tribenuron-

methyl in flixweed (Descurainia sophia L.). BMC Genomics 17: 1-13.

Yuan, J. S., Tranel, P. J., Stewart, C. N. 2007. Non-target-site herbicide resistance: a family

business. Trends in Plant Science 12: 6-13.

11

CHAPTER II

REVIEW OF LITERATURE

12

Review of Literature

Palmer amaranth

Palmer amaranth (Amaranthus palmeri S Watson, subgenus Acnida, subsection

Saueranthus) is one of the most common and problematic weed species in corn (Zea mays),

cotton (Gossypium hirsutum), grain sorghum (Sorghum bicolor), peanut (Arachis hypogea), and

soybean (Glycine max) in the US. It is described as a tall (usually about 2.5 m tall), erect,

branched summer annual plant with leaves arranged alternately on the stems. The pistillate and

staminate flowers occur in separate plants (dioecious species) in long, narrowly elongated

terminal inflorescence. The female inflorescences have sharp bracts which are distinct from male

inflorescences, which are soft to touch. Because it is a dioecious species, Palmer amaranth is an

obligate outcrosser (Ward et al., 2013). Palmer amaranth can also hybridize, to a limited extent,

with spiny amaranth, which increases the risk of spreading herbicide resistance to other

Amaranthus species (Gaines et al., 2012).

Palmer amaranth is a C4 dicot with high rates of photosynthesis (81 μmol m-2 s-1)

compared to other C4 monocots such as corn and C3 dicots like cotton and soybean (Ehleringer,

1983; Gibson, 1998). Under ideal conditions, Palmer amaranth can grow up to 5 cm daily.

(Horak and Loughin, 2000). Temperature plays an important role in the net rate of

photosynthesis in Palmer amaranth, and optimum photosynthesis occurs between 36-46°C

(Ehleringer, 1983). Furthermore, Palmer amaranth exhibits diaheliotropism, meaning the plant is

capable of tracking sunlight where the leaves orient themselves perpendicularly to the sun rays to

maximize light interception and thus photosynthesis (Ehleringer and Forseth, 1980).

Infestation of Palmer amaranth significantly affects growth and yield of most agronomic

crops, cotton being one of the most sensitive commodities. Cotton yield decreased by 5.38% for

13

every one additional Palmer amaranth plant per 9.1 m of row (Morgan et al., 2001). Palmer

amaranth interference can cause significant yield reduction in soybean (Bensch et al., 2003), corn

(Massinga et al., 2001), peanut (Burke et al., 2007), and sweet potato (Ipomoea batatas) (Meyers

et al., 2010) crop productions. The success of Palmer amaranth as troublesome weed is attributed

to its extended emergence period (Jha and Norsworthy, 2009), high fecundity (Keeley et al.,

1987), rapid growth rate (Jha et al., 2008) with a C4 photosynthetic pathway (Ehleringer, 1983),

genetic diversity, ability to tolerate adverse conditions, and its facility for evolving herbicide

resistance (Ward et al., 2013).

Palmer amaranth can be controlled by timely applications of preemergence (PRE) and

postemergence (POST) herbicides. Some of the PRE herbicides labeled on different crops for

Palmer amaranth control are diuron (e.g. corn, cotton, grain sorghum), fluometuron (e.g. cotton,

sugarcane), fomesafen (e.g. soybean, cotton), metribuzin (e.g. soybean), pendimethalin (e.g.

corn, cotton, soybean, and many other crops), pyrithiobac and trifloxysulfuron(cotton),

pyroxasulfone (e.g. corn, cotton, soybean, wheat), saflufenacil ( e.g. corn, cotton, alfalfa), S-

metolachlor (e.g. corn, cotton, soybean, and many other crops), and tembotrione (e.g. corn)

(Scott et al. 2017). Labelled post-emergence herbicides for Palmer amaranth control include

atrazine (e.g. corn, sorghum), dicamba (e.g. corn, sorghum), fomesafen (e.g. soybean),

glufosinate (e.g. LibertyLink® crops), glyphosate (e.g. Roundup Ready® crops), and mesotrione

(e.g. corn, sorghum) (Norsworthy et al., 2008).

The problem of Palmer amaranth escalated with the evolution of herbicide-resistant

populations due to intensive use of herbicides. As a dioecious species, Palmer amaranth is

outcrossing, allowing herbicide resistance to spread rapidly (Steckel, 2007). The glyphosate

resistance trait was transferred across a 300-m distance through pollen from glyphosate-resistant

14

males to glyphosate-susceptible female plants (Sosnoskie et al., 2012). Resistance to six different

modes of action (MOA) has been documented in Palmer amaranth: acetolactate synthase (ALS)

inhibitors, dinitroanilines, triazine, glyphosate, hydroxyphenylpyruvate dioxygenase (HPPD)

inhibitors, and protoporphyrinogen oxidase (PPO) inhibitors (Heap 2018). Some Palmer

amaranth populations are resistant to more than one MOA (Burgos et al., 2001). The rampant

occurrence of ALS- and glyphosate-resistant Palmer amaranth populations has raised great

challenge to farmers and researchers as this curtails the use of ALS inhibitors and glyphosate in

crop production systems. With glyphosate and ALS inhibitors out of the weed management

toolbox, weed control has returned to multiple-herbicide programs including PSII, PPO, and

shoot/root inhibitors. Genetically modified crops with tolerance to 2,4-D and dicamba are

recently commercialized and new technologies on HPPD-tolerant soybean (mesotrione and

isoxaflutole) will be available soon. These alternative modes of action should be wisely used to

preserve the utility of these herbicides and delay the evolution of resistant weeds.

Non-target-site-based resistance mechanism

There are two primary mechanisms of herbicide resistance in weeds: 1) target-site

resistance (TSR) which is caused by alterations in the gene of the herbicide target site causing a

decrease in the affinity of the herbicide for its target and 2) non-target-site resistance (NTSR)

which is endowed by mechanisms not belonging to TSR (Delye, 2013). NTSR mechanisms

include mechanisms that minimize the amount of herbicide in reaching the target site such as

decreased herbicide uptake and translocation, increases herbicide sequestration, and metabolism

(Powles and Yu, 2010). NTSR can confer unpredictable resistance to multiple herbicides with

different MOA, including herbicides not yet used (Petit et al., 2010a; Petit et al., 2010b).

15

Sublethal dose of herbicide has been implicated to promote non-target-site metabolic resistance

(Neve and Powles, 2005; Yuan et al., 2007). NTSR within a single plant is generally endowed by

a combination of mechanisms controlled by different alleles as demonstrated in the segregation

analysis of NTSR to ACCase ad ALS herbicides in progenies from controlled pairings of black-

grass (Alopecurus myosuroides) parents (Petit et al., 2010b).

NTSR mechanisms as a part of plant stress response can interfere with herbicide

penetration and accumulation at the target site, and/or protect the plant against the phytotoxic

effects of herbicide action. Detoxification of herbicide usually follow a three-step process: 1)

transformation of the herbicide molecule into a more hydrophilic metabolite by P450

monooxygenase, 2) conjugation of metabolites to glutathione or glucose, 3) further conjugation,

cleaving and oxidizing and export of the molecule to the vacuole or cell wall for final

degradation (Delye, 2013). Increased expression and activity of the enzymes that are involved in

the detoxification process can contribute to reduced sensitivity of the plant to the herbicide.

Reduced herbicide translocation may also be connected with detoxification of herbicides.

Reduced translocation of glyphosate in horseweed (Conyza candensis) and ryegrass (Lolium spp)

is due to rapid sequestration of glyphosate into the vacuole (Ge et al., 2012; Ge et al., 2010).

Reduced herbicide absorption in an ACCase-resistant rigid ryegrass (Lolium rigidum) biotype is

due to greater epicuticular wax density in the leaf cuticle (de Prado et al., 2005).

The basis for crop-weed selectivity for many herbicides is due to differential herbicide

metabolism. The herbicide is safe for the crop because the crop metabolizes the herbicide

whereas the weed does not. Enhanced herbicide metabolism is more common in grass weeds, but

additional cases involving dicot weeds are emerging (Yu and Powles, 2014). The co-application

of malathion, a P450 inhibitor, resulted in loss or decreased level of herbicide resistance in

16

several weed species demonstrated that enhanced herbicide metabolism is attributed to the

cytochrome P450 family of proteins (Beckie et al., 2012; Breccia et al., 2017; Fu et al., 2017;

Zhao et al., 2017). An enhanced herbicide metabolism has been reported in rigid ryegrass (Yu et

al., 2013), wild mustard (Sinapis arvensis) (Veldhuis et al., 2000a), and late watergrass

(Echinochloa phyllopogon) (Yasuor et al., 2009; Yun et al., 2005). Cytochrome P450s have been

implicated in the metabolism of different herbicides in several crops and weed species

(Cotterman and Saari, 1992; Hinz and Owen, 1996; Yu and Powles, 2014). The cloning of rice

(Oryza sativa) CYP81A6 and Jerusalem artichoke (Helianthus tuberosus) CYP76B1 into

Arabidopsis conferred resistance to sulfonylurea and phenylurea herbicides, respectively (Pan et

al, 2006; Liu et al. 2012; Didierjean et al. 2002).

In addition to P450, glutathione S-transferases (GSTs) and glycosyltransferases have

been implicated in herbicide metabolism (Yuan et al., 2007). Each of these three enzyme systems

is encoded by large gene families. GSTs are ubiquitous in plants and have defined roles in

herbicide detoxification (Wagner et al., 2002). Maize (Zea mays)and giant foxtail (Setaria

faberi) are tolerant to atrazine due to its ability to rapidly detoxify the herbicide atrazine by

glutathione conjugation with GST (Hatton et al., 1999; Hatton et al., 1996). Furthermore,

induction of glutathione and GSTs by herbicide 'safeners' is employed commercially to protect

crop plants from injury by certain herbicides (Farago et al., 1994). GSTs (phi and tau class) were

identified to be involved in diclofop resistance in rigid ryegrass (Gaines et al., 2014).

Furthermore, GSTA and GST1 were also recently reported to participate in non-target resistance

or tolerance to ALS inhibitor in ryegrass and maize, respectively (Duhoux et al., 2015; Liu et al.,

2015). A transgenic tobacco plant (Nicotiana tabacum) expressing maize GST1 had enhanced

detoxification of alachlor (Karavangeli et al., 2005). In ACCase-resistant black-grass, GSTF1

17

catalyzed the conjugation of herbicide to glutathione and acted as peroxidases protecting the cell

from oxidative damage (Cummins et al., 2009; Cummins et al., 1999; Cummins et al., 2013).

Transgenic Arabidopsis expressing the black-grass GSTF1gene (AmGSTF1) has improved

tolerance to some herbicides due to increased accumulation of protective flavonoids (Cummins

et al., 2013).

Few non-target mechanisms have been elucidated at the molecular level because of the

inherently complicated biochemical processes and limited genomic information available in

weeds species (Yuan et al., 2007). Although NTSR mechanisms are widespread among weed

species, its genetic determinant is poorly understood (Delye, 2013). The de novo transcriptome

for waterhemp (Amaranthus tuberculatus) was characterized using GS-FLX 454 sequencing

which can help in establishing valuable genomic resource for weed science research (Riggins et

al., 2010). The transcriptome is useful for identifying potential candidate genes involved

herbicide resistance and may serve as a reference for gene expression and functional genomics

studies Using whole-transcriptome sequencing (RNA-Seq), several genes were identified in

ryegrass, black-grass, flixweed (Descurainia sophia L.), and shortawn foxtail (Alopecurus

aequalis) that confer NTSR to several herbicides (Gaines et al. 2014; Duhoux et al. 2017; Gardin

et al. 2015; Yang et al. 2016; Zhao et al. 2017). Identifying NTSR genes will help in

understanding NTSR evolution as well as help in understanding weed adaptation to herbicides.

Glufosinate

Glufosinate is a broad-spectrum, non-selective, post-emergence herbicide commonly

used to control weeds in vineyards, orchards and genetically modified crops with LibertyLink®

trait. Glufosinate inhibits glutamine synthetase (GS) (E. C. 6.3.1.2) enzyme which catalyzes the

18

conversion of glutamic acid and ammonia to form glutamine, the first reaction in the ammonia

assimilation in higher plants. Glutamine synthetase functions as the major assimilatory enzyme

for ammonia produced from nitrogen fixation, and nitrate or ammonia nutrition, as well as

reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and

nitrogen transport compounds (Miflin and Habash, 2002). Inhibition of GS enzyme leads to rapid

ammonia accumulation and amino acid biosynthesis inhibition which cause damage in the

chloroplast structures and terminates photosynthetic activity, ultimately leading to plant death

(Tachibana et al., 1986). The availability of glufosinate-tolerant cotton, soybean, and corn

varieties allows farmers to use glufosinate in controlling broad spectrum of weeds without

damaging the crop as well as provides growers an alternative herbicide for controlling Palmer

amaranth that is resistant to other herbicides.

Despite the non-selective nature of glufosinate, variable control of goosegrass (Eleusine

indica), large crabgrass (Digitaria sanguinalis) and Palmer amaranth has been observed (Beyers

et al., 2002; Corbett et al., 2004; Everman et al., 2007). Differential tolerance to glufosinate in

weed species has been attributed to application rate, plant species, application timing, humidity,

growth stage, and variations in the level of absorption and translocation (Everman, 2007).

Resistance to glufosinate has been reported in goosegrass (Seng et al., 2010) and Italian ryegrass

(Avila-Garcia and Mallory-Smith, 2011). In glufosinate-resistant Italian ryegrass (Lolium

perenne L. ssp multiflorum) population from Oregon, resistance is attributed to mutation of the

GS gene in which aspartic acid was substituted to asparagine at the amino acid position 171

(Avila-Garcia et al., 2012). The overproduction of GS enzyme conferred resistance to glufosinate

in rice lines (Tsai et al., 2006). Glufosinate-tolerant crops are developed by expressing

phosphinothricin N-acetyltransferase (PAT), which can detoxify L-phosphinothricin

19

(glufosinate) by acetylation of the amino group (Droge et al. 1992). Although resistance to

glufosinate in Palmer amaranth has not been reported, tolerance to glufosinate has been observed

in a population of Palmer amaranth in Arkansas (Botha, 2012).

ALS-inhibiting herbicides

Acetolactate synthase, also known as acetohydroxyacid synthase or AHAS (E.C.

4.1.3.18), is the first enzyme in the biosynthesis of the branched chain amino acids Ile, Leu, and

Val. Inhibition of ALS leads to depletion of these amino acids disrupting protein synthesis,

thereby causing plant death (Whitcomb, 1999). There are five chemical families of ALS

herbicides, namely: sulfonylurea (SU), imidazolinone (IMI), triazolopyrimidine sulfonanilides

(TP), pyrimidinylthiobenzoates (PTB), and sulfonylaminocarbonyltriazolinone. The ALS

herbicides were widely used because they provide broad-spectrum weed control at low doses,

soil residual activity, wide application windows, excellent crop safety and low mammalian

toxicity. However, these herbicides have high propensity to select resistant weed populations due

to its widespread usage, strong selection pressure, and high levels of natural variability in the

ALS gene. Selection of ALS-resistant weed populations became evident in 1987, only five years

after the introduction of the first SU, with the discovery of chlorsulfuron-resistant prickly lettuce

(Lactuca serriola L.) and kochia (Kochia scoparia L. Shrad) (Mallory-Smith et al., 1990;

Primiani et al., 1990). In the United States, the first case of resistance to ALS-inhibiting

herbicides in Palmer amaranth was reported in 1993 in Kansas and cross-resistance to five ALS

herbicides was documented in Arkansas in 1994 (Heap 2018). Resistance to ALS inhibitors in

Palmer amaranth is widespread throughout the United States. As a result, ALS-inhibiting

herbicides are no longer effective on ALS-resistant populations of Palmer amaranth.

20

Most cases of ALS resistance are due to mutation in the ALS gene resulting in an enzyme

that is less sensitive to herbicide binding. To date, 29 amino acid substitutions in ALS conferring

herbicide resistance were identified at Ala122 (5), Ala205 (2), Arg377 (1), Asp376 (1), Gly654 (2),

Pro197 (11), Ser653 (3), and Trp574(4) in weed species (Heap 2018). Recent investigation on 20

Palmer amaranth populations from Arkansas revealed TSR mechanism involving Trp574Ser

mutation with a few cases of double mutations involving Ala122Thr, Pro197Ala or Ser653Asn

(Singh, 2017). Amino acid substitution at Pro197 conferred resistance to SUs with low or no

cross-resistance to IMIs (Tranel and Wright, 2002), whereas substitutions at Ala122 or Ser653

conferred resistance to IMI herbicides with low-level resistance to SUs (Bernasconi et al., 1996;

Devine et al., 1991). Substitution at Trp574 confers resistance to IMIs, PTBs, SUs, and TPs

(Tranel and Wright, 2002), whereas Asp376Gluand Ala205Phe substitution confers resistance to all

five chemical families of ALS inhibitors (Brosnan et al., 2016; Whaley et al., 2007).

An important mechanism of naturally occurring (as opposed to evolved) resistance to

ALS inhibitors is detoxification of the active herbicide in the plant. Inherent selectivity of a

particular ALS inhibitor in a given crop is based on the crops’ ability to metabolize the herbicide

to nonphytotoxic compounds rapidly enough to prevent lethal herbicide levels from reaching the

target enzyme ALS (Saari et al. 1994). Among the more common detoxification reactions

involved in crop tolerance to sulfonylureas are hydroxylation, O-dealkylation, and

deesterification (Saari et al. 1994). Maize is tolerant to nicosulfuron, a sulfonylurea herbicide,

because nicosulfuron is rapidly metabolized to 5-hydroxypyrimidinyl nicosulfuron, a

herbicidally inactive derivative which is then conjugated to glucose (Brown et al. 1991).

Similarly, flumetsulam, a triazolopyrimidine that is selective in cereals, maize, and soybeans,

also owes its selectivity to metabolic detoxification. Tolerant plants oxidize flumetsulam to one

21

or more hydroxylated metabolites, and soybean produces an open pyrimidine ring metabolite

(Swisher et al., 1991). This tolerance mechanism in crops also appears to be the same mechanism

responsible for poor control of some weeds by certain ALS herbicides (Saari et al. 1994).

Non-target-site resistance to ALS inhibitors have been reported in some weeds species.

Reduced absorption and translocation rarely underlay resistance to ALS inhibitors (Cruz-

Hipolito et al., 2013; Poston et al., 2001; Veldhuis et al., 2000b) and in only few cases have they

been reported as partial-resistance mechanism (Riar et al., 2013; White et al., 2002) . An

enhanced herbicide metabolism has been reported in rigid ryegrass (Yu et al., 2013), wild

mustard (Veldhuis et al., 2000a), and late watergrass (Yasuor et al., 2009; Yun et al., 2005). A

late watergrass biotype with multiple herbicide resistance to bispyribac-sodium, fenoxaprop-

ethyl, and thiobencarb exhibited higher P450 hydroxylation activity toward these herbicides than

the susceptible biotype, which suggests the involvement of cytochrome P450 enzymes as a

mechanism for resistance (Yun et al. 2005). Furthermore, resistance to penoxsulam in late

watergrass is conferred by an enhanced ability to detoxify the herbicide via malathion-sensitive

monooxygenases (Yasour et al. 2009). The P450 inhibitor malathion reverses chlorsulfuron

resistance in rigid ryegrass (Yu et al. 2009). Several cytochrome P450 have been identified in

ALS-resistant weeds, such as CYP72A254 (also known as CYPA72A219) in late watergrass

(bispyribac-sodium) (Iwakami et al., 2014), CYP81D in black-grass (mesosulfuron and

iodosulfuron) (Gardin et al., 2015); CYP94A1, CYP94A2, CYP71A4 and CYP734A6 in

shortawn foxtail (mesosulfuron) (Zhao et al. 2017). In addition, many CytP450s conferring

resistance to various herbicides have been documented in several major crop species, such as

CYP81A6 in rice (Pan et al., 2006); CYP76B1 in Jerusalem artichoke (Didierjean et al., 2002),

and CYP81A9 in maize (Zea mays) (Liu et al. 2015). The wheat (Triticum aestivum)

22

CYP71C6v1 expressed in yeast (Saccharomyces cerevisiae) is able to metabolize several

sulfonylurea herbicides such chlorsulfuron and triasulfuron through phenyl ring hydroxylation

(Xiang et al., 2006).

PPO-inhibiting herbicides

Herbicides that inhibit protoporphyrinogen oxidase (PPO) include lactofen and

fomesafen herbicides. Although they have been used for many years in the control of broadleaf

weeds, their use began to slowly decline in the late 1990s due to the massive adoption of

glyphosate-resistant varieties (Riggins et al., 2010). However, the advent and continued increase

of glyphosate-resistant weeds cause growers to once again rely on PPO herbicides as an

alternative approach to control weeds. Even though resistance to PPO herbicide has been slow to

evolve, it may be expected to occur in weed species that are under strong and continuous

selection pressure.

The PPO enzyme plays a major role in the biosynthesis of chlorophyll and heme by

catalyzing the oxidation of protoporphyrinogen (protogen) to porphyrin IX (Proto IX). Upon

treatment with PPO-herbicides, susceptible plants accumulate protogen IX which is then

transported to the cytoplasm where it spontaneously form Proto IX. In presence of light, Proto IX

generates singlet oxygen that causes degradation of cell membranes leading cells to break open

and ultimately to cellular death (Becerril and Duke, 1989; Jacobs et al., 1991; Sherman et al.,

1991). However, few species of weeds have slowly evolved resistance from the repeated use of

these herbicides in past few years. To, date 11 different weed species have evolved resistance to

PPO-herbicides including Palmer amaranth (Heap 2018; Giacomini et al., 2017; Salas et al.,

2016; Salas-Perez et al., 2017).

23

The resistance mechanism to PPO herbicides in tall waterhemp is not due to difference in

herbicide absorption and translocation, or metabolism (Shoup and Al-Khatib, 2005) but due to a

unique codon deletion of glycine at position 210 in the mitochondrial isoform of the PPO2 gene

(Patzoldt et al., 2006). The same mutation appears to be the only resistance mechanism to PPO

herbicides in various tall waterhemp populations in the United States (Lee et al., 2008; Wuerffel

et al., 2015). This glycine deletion alters the binding domain of the enzyme without negatively

affecting substrate affinity (Dayan et al., 2010). However, a novel PPO mutation, Arg98Leu was

detected in common ragweed (Ambrosia artemisiifolia), and recently two new substitutions

identified at the same position in Palmer amaranth as Arg128Gly and Arg128Met (numbering

changed due to the presence of 30 amino acid extension in Palmer amaranth) (Giacomini et al.,

2014; Rousonelos et al., 2012). Computational modeling revealed that replacement of this

arginine with hydrophobic glycine removed important hydrogen-bonding interactions with

acifluorfen, fomesafen, and sulfentrazone resulting in reduced binding of these inhibitors (Hao et

al., 2014).

Non-target resistance to PPO inhibitors in weed species has not yet been reported

although natural tolerance to PPO inhibitors in crops is usually due to enhanced herbicide

degradation. Diphenyl ether herbicides are detoxified in soybean by homoglutathione

conjugation (Skipsey et al., 1997). Similarly, tolerance to the diphenyl ether fluorodifen in peas

is due to rapid conjugation with glutathione (Edwards, 1996).

24

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31

CHAPTER III

RNA-SEQ TRANSCRIPTOME ANALYSIS OF AMARANTHUS PALMERI WITH

DIFFERENTIAL TOLERANCE TO GLUFOSINATE HERBICIDE

32

Abstract

Amaranthus palmeri (Amaranthaceae) is a noxious weed in several agroecosystems and in

some cases seriously threatens the sustainability of crop production in North America.

Glyphosate-resistant Amaranthus species are widespread, prompting the use of alternatives to

glyphosate such as glufosinate, in conjunction with glufosinate-resistant crop cultivars, to help

control glyphosate-resistant weeds. An experiment was conducted to analyze the transcriptome

of A. palmeri plants that survived exposure to 0.55 kg ha-1 glufosinate. Since there was no record

of glufosinate use at the collection site, survival of plants within the population are likely due to

genetic expression that pre-dates selection; in the formal parlance of weed science this is

described as natural tolerance. Leaf tissues from glufosinate-treated and non-treated seedlings

were harvested 24 h after treatment (HAT) for RNA-Seq analysis. Global gene expression was

measured using Illumina DNA sequence reads from non-treated and treated surviving

(presumably tolerant, T) and susceptible (S) plants. The same plants were used to determine the

mechanisms conferring differential tolerance to glufosinate. The S plants accumulated twice as

much ammonia as did the T plants, 24 HAT. The relative copy number of the glufosinate target

gene GS2 did not differ between T and S plants, with 1 to 3 GS2 copies in both biotypes. A

reference cDNA transcriptome consisting of 72,780 contigs was assembled, with 65,282

sequences putatively annotated. Sequences of GS2 from the transcriptome assembly did not

have polymorphisms unique to the tolerant plants. Five hundred sixty-seven genes were

differentially expressed between treated T and S plants. Of the upregulated genes in treated T

plants, 210 were more highly induced than were the upregulated genes in the treated S plants.

Glufosinate-tolerant plants had greater induction of ABC transporter, glutathione S-transferase

(GST), NAC transcription factor, nitronate monooxygenase (NMO), chitin elicitor receptor

33

kinase (CERK1), heat shock protein 83, ethylene transcription factor, heat stress transcription

factor, NADH-ubiquinone oxidoreductase, ABA 8’-hydroxylase, and cytochrome P450 genes

(CYP72A, CYP94A1). Seven candidate genes were selected for validation using quantitative real

time-PCR. While GST was upregulated in treated tolerant plants in at least one population,

CYP72A219 was consistently highly expressed in all treated tolerant biotypes. These genes are

candidates for contributing tolerance to glufosinate. Taken together, differential induction of

stress-protection genes in a population can enable some individuals to survive herbicide

application. Elevated expression of detoxification-related genes can get fixed in a population

with sustained selection pressure, leading to evolution of resistance. Alternatively, sustained

selection pressure could select for mutation(s) in the GS2 gene with the same consequence.

34

Introduction

Amaranthus palmeri is a dioecious, weedy Amaranthus species native to Southwestern North

America [1, 2]. It is one of the most widespread, troublesome, and economically damaging

weeds in agronomic crops throughout the southern United States [2]. Infestation of Palmer

amaranth can cause from 70% to more than 90% yield loss in cotton (Gossypium hirsutum) [3],

soybean (Glycine max) [4], and corn (Zea mays) [5]. A. palmeri is difficult to control because of

its rapid growth rate, high fecundity, tiny seeds dispersed by multiple agents, continuous

emergence pattern, high genetic diversity, high propensity for evolving herbicide resistance, and

dioecious nature with long-distance pollen dispersal [1, 6-9]. To date, resistances to six

herbicide mechanisms of action (MOAs) have been confirmed in A. palmeri: acetolactate

synthase (ALS) inhibitors, 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors,

enolpyruvyl shikimate-3-phosphate synthase (EPSPS) inhibitor (glyphosate), mitosis inhibitors

(dinitroanilines), photosystem II inhibitors (triazines), and protoporphyrinogen oxidase (PPO)

inhibitors [10]. The increasing resistance of A. palmeri to herbicides is a threat to corn, cotton,

peanut (Arachis hypogea), and soybean production [1, 11-14]. Alternative management

strategies are needed to combat this problem in several areas in North America [11, 15-18].

Herbicides are used as a major tool for controlling weeds and the evolution of herbicide-

resistant (HR) weeds is an increasing issue worldwide. Glyphosate-resistant (GR) crops, first

commercialized in 1996, were adopted quickly by growers because the technology allowed the

use of the nonselective herbicide, glyphosate, in-season. The technology drastically simplified

weed control with the use of a single, inexpensive, highly effective herbicide. In fact, GR crops

constituted 80% of the 175 million ha planted with transgenic crops globally [19]. However, the

over-reliance on glyphosate and its application over a vast land area has exerted unprecedented

35

selection pressure on weeds, resulting in the evolution of GR weeds including A. palmeri.

Glyphosate-resistant A. palmeri was first identified in Georgia in 2004 [20] and subsequently

became widespread across the South, Midwest and Northeast regions of the United States [10].

The widespread distribution of glyphosate-resistant weeds compelled farmers to use alternative

herbicides including another non-selective herbicide, glufosinate, to control HR weeds in

glufosinate-tolerant crops. Glufosinate is a fast-acting postemergence herbicide that controls

weeds by inhibiting glutamine synthetase (GS) (E.C. 6.3.1.2), the enzyme that converts

glutamate and ammonia to glutamine [21]. Inhibition of GS by glufosinate leads to ammonia

accumulation, inhibition of amino acid synthesis, and indirect inhibition of photosynthesis,

ultimately leading to plant death [22]. To date, resistance to glufosinate has been confirmed in

Eleusine indica from Malaysia [23, 24] and Lolium perenne ssp. multiflorum from Oregon [25].

An amino acid mutation in the chloroplast-encoded GS gene, Asp171Asn, conferred resistance to

glufosinate in L. perenne ssp. multiflorum [26]. Resistance to glufosinate in glufosinate-resistant

crops is achieved using transgenic methods to insert the bar or pat (phosphinothricin

acetyltransferase) gene from a bacterium to the plant’s genome, allowing detoxification of

glufosinate by acetylation [27].

Differential responses to glufosinate in weeds have been attributed to several factors

including light, temperature, humidity, growth stage, application rate, application timing,

species, and variation in the level of herbicide absorption, translocation, and metabolism [28,

29]. Control of A. palmeri by glufosinate is variable [30-32]. A previous study reported higher

uptake, mobility, and metabolism of glufosinate in A. palmeri compared to the more susceptible

Ipomoea lacunosa [28]. As is commonly observed, differential tolerance to herbicides are often

due to non-target-site (NTS) mechanisms, involving the detoxification of herbicide by

36

biochemical modification and/or the compartmentation of the herbicide and its metabolites [33].

Cases of weed resistance to herbicides due to NTS mechanisms have been increasing

(www.weedscience.org). The genetic bases of NTS mechanisms are not fully understood due to

the complex interaction of biochemical processes and limited genomic information on weedy

species [33-35]. In this study, we investigated A. palmeri accessions with differential tolerance to

glufosinate.

The genome of A. palmeri is not yet deciphered although the genome and transcriptome of its

cultivated relative grain amaranth (Amaranthus hypochondriacus) was completed in 2014 [36].

Also recently, the transcriptome of two weedy species Lolium rigidum [37] and Echinochloa

crus-galli [38] were sequenced to identify genes involved in herbicide resistance. Understanding

the molecular mechanisms underlying herbicide resistance could be used to mitigate and manage

resistance evolution and reduce weed impact on crops. This study assembled the transcriptome

sequence of A. palmeri exposed to glufosinate compared to controls to elucidate candidate genes

involved in differential tolerance to glufosinate.

Materials and Methods

Plant Materials

Amaranthus palmeri seed samples from 120 fields were collected in Arkansas, USA between

2008 and 2014. The collection of plant samples from the field was done with permission of farm

owners, managers, consultants, or Extension Agents. In the process of collecting samples, no

endangered species were affected. Inflorescences of at least 10 female plants per field were

harvested, dried, threshed, and cleaned for herbicide bioassays in the greenhouse. One accession

37

of interest (08-Lee-C) was collected from a field that was planted with glyphosate-tolerant

(Roundup Ready®) soybean in 2008 and glyphosate-tolerant cotton in 2006 and 2007. Although

this field had no record of being sprayed with glufosinate, some plants survived exposure to

glufosinate (0.55 kg ha-1) in the greenhouse. The survivors were grown and allowed to cross-

pollinate to produce the first cycle of purified (intercrossed) progeny (C1).

To study the potential survival mechanisms, seeds of 08-Lee-C and the C1 progeny were

planted in 4-cm-diameter pots using commercial potting soil mix (Sunshine Mix, Bellevue, WA,

USA). Seedlings (100) were grown at one plant per pot in a growth chamber maintained at 32/26

°C day/night temperature with a 16-h photoperiod. Plants were watered daily and fertilized with

a water-soluble all-purpose plant food containing 15-30-15% NPK (Miracle-Gro®, Marysville,

OH, USA), every 2 wk. Fifty plants per accession (9-cm tall) were sprayed with glufosinate

(0.55 kg ai ha-1) (Liberty®, Bayer) mixed with 20 g L-1 ammonium sulfate to identify S and T

plants. Susceptible and T plants from the non-treated control were identified by ammonia

accumulation assay. Six confirmed S plants from 08-Lee-C and T plants from C1 were used for

ammonia accumulation assay, determination of chloroplast-encoded glutamine synthetase (GS2)

copy number, and RNA-Seq experiment.

Phenotypic evaluation of A. palmeri response to glufosinate

The response of A. palmeri, collected between 2008 and 2014, was evaluated in the

greenhouse. A known herbicide-susceptible accession (SS) was included in each experiment as

control [39]. Five-hundred mg of seeds from each field-collected plant were mixed to make a

composite seed sample representing each accession. The experiment was conducted twice in a

randomized complete block design with two replications. Each replication consisted of one

cellular tray (28 X 54 cm) with 50 cells (Redway Feed Garden and Pet Supply, Reedway, CA,

38

USA) filled with a commercial medium (Sunshine Mix, Bellevue, WA). Composite seeds from

each accession were planted in each cell and seedlings were thinned to one per cell. Glufosinate

was applied at 0.55 kg ha-1 when seedlings were 7.5- 9 cm tall using a laboratory sprayer fitted

with a flat fan nozzle tip (800067 TeeJet, Spraying Systems Co., Wheaton, IL, USA) delivering

187 L ha-1 at 269 kPa. The herbicide was applied with 20 g L-1 ammonium sulfate. The plants

were assessed visually relative to the non-treated control 21 d after treatment (21 DAT) using a

scale of 0 to 100, where 0 = no visible injury and 100% = complete desiccation. The number of

survivors was recorded. Survivors from glufosinate treatment were grown to produce seed. Data

were analyzed using hierarchal clustering in JMP Pro v. 12.

Herbicide resistance profiling of a selected A. palmeri accession

Data from the differential tolerance evaluation were used to select an accession for further

study. Accession 08-Lee-C had the most number of survivors with minimum injury. Seeds from

08-Lee-C and SS accessions were planted as described in the previous section. Seedlings (7.5-9

cm tall) were treated with the recommended dose of fomesafen (264 g ha-1) (Flexstar®,

Syngenta), glyphosate (870 g ha-1) (Roundup PowerMAX®, Monsanto), dicamba (280 g ha-1)

(Clarity®, BASF), and ALS inhibitors pyrithiobac (73 g ha-1) (Staple LX®, DuPont) and

trifloxysulfuron (8 g ha-1) (Envoke®, Syngenta). The ALS inhibitors were applied with 0.25%

non-ionic surfactant (Induce®, Helena Chemical Co., Collierville, TN, USA), respectively.

Herbicide treatments were applied as described in the previous section. Mortality was evaluated

21 d after treatment. The experiment was conducted in a randomized complete block design as in

the previous section. Data were analyzed using ANOVA in JMP Pro v. 12.

39

Evaluation of tolerance level to Glufosinate

A dose-response bioassay was conducted in the greenhouse to determine the tolerance level

of 08-Lee-C and C1 relative to the SS accession to glufosinate. Seeds were sown in 15-cm

diameter pots filled with commercial potting soil. Seedlings, 7.5-cm tall, were sprayed with 11

doses of glufosinate using a laboratory sprayer as described in the previous section. The 08-Lee-

C and C1 accessions were sprayed with glufosinate from 0.0012 to 0.5940 kg ai ha-1, whereas the

SS accession was sprayed at 0.0006 to 0.5950 kg ha-1, with a non-treated check. The herbicide

was applied with 20 g L-1 ammonium sulfate. Shoot biomass was harvested 21 DAT, dried at

60°C for 72 h, and weighed. The experiment was conducted in a randomized complete block

design with four replications. Five plants were used per replication (20 plants total) per herbicide

concentration.

Data were analyzed using SAS JMP Pro v. 13 in conjunction with SigmaPlot v.13 (Systat

Software, Inc., San Jose, CA, USA) for nonlinear regression analysis. The percentage biomass

reduction was fitted to a nonlinear, sigmoid, four-parameter logistic regression model defined by

𝑦 = 𝑐 + [(𝑑 − 𝑐)/(1 + 𝑒{−𝑎(𝑥 − 𝑏)})]

where y represents the biomass reduction expressed as percentage relative to the non-treated

control, a is the growth rate, b is the inflection point, c is the lower asymptote, d is the upper

asymptote, and x is the glufosinate dose. The herbicide doses that would cause 50% growth

reduction (GR50) were estimated using the fitted regression equation.

Ammonia accumulation assay

To identify S and T plants without glufosinate treatment, a leaf disc assay was conducted to

measure ammonia accumulation caused by the inhibition of photorespiration by glufosinate [40].

40

The assay was conducted using 50 non-treated plants each from 08-Lee-C and the C1 progeny.

In addition, leaf tissues from three 08-Lee-C plants that were controlled (S) and three C1 plants

that survived (T) glufosinate application at the whole plant level were also tested. From each

plant, two leaf discs (5-mm diameter) were excised from the youngest, fully expanded leaf of

6.4-cm tall seedlings. One leaf disc was placed per well in a microtiter plate containing 200 µM

glufosinate. The plate was sealed with micropore tape and placed on a bench under light for 24 h.

The plate was moved to a -80 °C freezer to stop the reaction. After two freeze-thaw cycles,

ammonia content was measured in a spectrophotometer (Pharma Spec UV-1700, Shimadzu,

Columbia, MD) at 630 nm using a modified method by Molin and Khan [41]. Leaf discs from S

plants were expected to have higher ammonia content than those from T plants.

Glutamine synthetase (GS2) relative copy number

Leaf tissues were harvested from confirmed S and T plants (three each) without glufosinate

treatment and stored at -80°C until processing. Leaf tissues were harvested also from plants

treated with glufosinate, 24 HAT. Upon evaluation of plant response 21 DAT, leaf tissues from

three S and T plants were used also to determine the relative copy number of GS2 gene. Genomic

DNA was extracted using the modified hexadecyltrimethylammonium bromide (CTAB) method

[42]. Quantitative real-time PCR (qPCR) was used to determine the genomic copy number of

GS2 relative to a housekeeping RNA dead box helicase gene GS2 in A. palmeri. The primer pair

GS2-F (5’- ATACGGAGAAGGAAGGCAAAG -3’) and GS2-R (5’-

TGTGGGTTCCCAAAGTAGTG-3’) were designed to amplify a region of the chloroplast GS.

RNA dead box helicase gene primers A36-F (5’- TTGGAACTGTCAGAGCAACC-3’) and A36-

R (5’-GAACCCACTTCCACCAAAAC-3’) were used as internal primers to normalize the

samples for any differences in DNA quantities. Reactions were conducted in three technical

41

replicates, and a negative control consisting of primer pairs with no template was included. An 8-

fold serial dilution of genomic DNA samples, ranging from 0.00064 to 50 ng, was used to

construct a standard curve. The slope of the standard curve was used to determine amplification

efficiency (E). The qPCR reaction efficiency was 97% with an R2 of 0.9907 and a slope of 3.271

indicating good assay validation. Genomic DNA templates (2 ng) were amplified in a 25-µL

reaction containing 12.5-µL Bio-Rad iQ SYBR Green Supermix, 2-µL of primers (1:1 mix of

forward and reverse primers), and nuclease-free water. Reaction conditions included 10 min

incubation at 94°C, then 40 cycles of 94°C for 15 s and 60°C for 1 min, followed by a melt-curve

analysis to confirm single PCR product amplification. Data were analyzed using CFX Manager

software (v.1.5). Relative GS2 copy number was calculated as ∆𝐶𝑡 = (𝐶𝑡, 𝐴36 − 𝐶𝑡, 𝐺𝑆2)

according to the method described by Gaines et al [43]. Increase in GS2 copy number was

expressed as 2ΔCt. Results were expressed as the fold increase in GS2 copy number relative to

RNA dead box helicase.

RNA-Seq Analysis

Sample preparation for RNA-Seq

This experiment used leaf tissues from non-treated and treated, confirmed S and T plants.

These were the same plants used for ammonia assay and GS2 copy number determination.

Tissues were collected 24 h after glufosinate application for RNA extraction. This collection

time was selected to capture herbicide stress adaptation genes and because maximum absorption

of glufosinate occurs 24 HAT [28]. Treatments were designated as non-treated S (susceptible

without treatment, SWO), non-treated tolerant (tolerant without treatment, TWO), treated

susceptible (SWT), and treated tolerant (TWT) plants with three biological replicates. Total RNA

42

was extracted from young leaf tissues of S and T plants using PureLink RNA Mini Kit (Life

Technologies, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted RNA

was treated with DNase (Invitrogen, Carlsbad, CA, USA) to remove potential genomic DNA

contamination, according to the manufacturer’s instructions. The samples were then sent to the

Clemson University Genomics and Computational Biology Laboratory, South Carolina for

sequencing the transcriptome.

Transcriptome sequencing and assembly

Total RNA was normalized and converted to cDNA using the TruSeq RNA library kit v2.0

(Illumina). Final sequencing products were validated for size on an Agilent Bioanalyzer 2100

(Agilent Technologies, Waldbronn, Germany) and sequenced using a 2x125bp paired-end

sequencing module on an Illumina HiSeq 2500 (Illumina). Raw sequence reads were assessed for

quality using the FastQC software package

(http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and preprocessed to remove

sequence adapters and low quality bases with the Trimmomatic software [44]. A reference

unigene assembly comprehensive of developmental stage, tissue source, and experimental

conditions was prepared by concatenating all preprocessed reads and assembling with the Trinity

software package [45]. The resulting unigene assembly was filtered for genuine coding

sequences (e.g., sequences without internal stop codons or chimeras) with the TransDecoder

software, and clustered by identity with the CD-HIT software [46] in an attempt to collapse

homologs, but not paralogs, at high identity thresholds.

Differential gene expression (DGE)

Paired-end reads from each individual were aligned to the de novo transcriptome using the

Subread package [47, 48]. Samtools was used to convert alignments from sam to bam format,

http://www.bioinformatics.babraham.ac.uk/projects/fastqc/

43

sort, and index [49, 50]. Subread’s featureCounts counted the number of reads that aligned to

each gene in the transcriptome [47, 48]. The final gene counts were loaded into Bioconductor’s

edgeR package for statistical analysis [51-55]. Variance between samples was visualized by a

multidimensional scaling (MDS) plot. Volcano plots were generated for each comparison of

samples. The criteria for differential gene expression included a fold-change >2 between

compared groups and statistical significance at P<0.05 [56]. Expression differences were

compared between non-treated T and non-treated S (TWO vs SWO), treated T and treated S

(TWT vs SWT), treated T and non-treated T (TWT vs TWO), and between treated S and non-

treated S (SWT vs SWO).

Transcriptome annotation

The final reference assembly was annotated by blastx and blastp alignment to the non-

redundant protein database at NCBI, the UniProt-swissprot database, and the uni-ref database to

determine homology to known genes. Protein domains were determined by alignment to the

HMMER database (http://hmmer.janelia.org). Signal peptides were determined with the SignalP

software [57] and transmembrane regions predicted with tmHMM

(http://www.cbs.dtu.dk/index.shtml). Gene ontology terms were derived from the best BLAST

match [58] and clustered to determine enrichment using the Agbase tool [59].

The entire dataset was submitted as NCBI BioProject (PRJNA390774), which is a part of the

U.S. National Library of Medicine of the National Institutes of Health. The 12 samples that were

used to construct the transcriptome, and to run the differential gene expression comparisons were

submitted as 12 separate BioSamples, SAMN07260017-SAMN07260028. The trimmed, paired-

end fastq files for each of the 12 samples were submitted to the Sequence Read Archive,

SRR5759376 – SRR5759387. Finally, the transcriptome was submitted to the Transcriptome

http://hmmer.janelia.org/

44

Shotgun Assembly Sequence Database. The transcriptome, consisting of 72,780 transcripts, is

under TSA submission: SUB2788796.

Sequence analysis of the glutamine synthetase 2 (GS2) gene

Glutamine synthetase 2 (GS2) gene sequences of the T and S biotypes were extracted and

assembled from the transcriptome data. A 1296-bp GS2 gene (431 amino acids) from S and T

plants was sequenced. The full length GS2 sequences of S and T plants were aligned using

Sequencher 5.4.6 and BioEdit software packages to identify amino acid substitutions. Sequence

alignment also included GS2 sequences of other Amaranthus species (A. viridis, A. albus, A.

spinosus, A. hybridus, A. lividus and A. thunbergii) available in the database at

http://www.weedscience.org.

Heat Map Analysis

Differentially expressed genes associated with abiotic stress response were subsampled and

subjected to heat map analysis. Normalized read count averages were calculated to produce

biological expression profiles followed by hierarchical clustering to recursively merge

expression based on pair-wise distances between non-treated T (TWO), non-treated S (SWO),

glufosinate-treated T (TWT), and glufosinate-treated S (SWT) samples. Digital expressions were

visualized between rows for normalized read count numbers (minimum & maximum) expression

patterns. The expression pattern was generated using GENE-E tool

(http://www.broadinstitute.org/cancer/software/GENE-E/).

Selection of candidate non-target genes

Genes that were commonly expressed between treated S and T, and between treated T and

non-treated T plants were selected for further evaluation based on their gene ontologies (GO).

https://urldefense.proofpoint.com/v2/url?u=http-3A__www.weedscience.org&d=DgMFaQ&c=7ypwAowFJ8v-mw8AB-SdSueVQgSDL4HiiSaLK01W8HA&r=ZVNBNfLtvpDwIUDWhIFjHg&m=gY_JxOefQsG1VfJ3qTEQmocNKaP_4eJNlSjXrczebQc&s=PZJMcS41wDhU7bm_PWvIzVUE9GDIW18PVUp9NpNsVsQ&e=

http://www.broadinstitute.org/cancer/software/GENE-E/

45

Genes that were assigned with GO molecular function and biological process related to

metabolism and signaling pathways (oxidoreductase activity, nuclear acid binding transcription

factor activity, hydrolase activity, transferase activity, transmembrane transporter activity,

transferase activity, protein transporter activity, biosynthetic process, small molecule metabolic

process, signal transduction, homeostatic process, immune system process, cell wall

organization, secondary metabolic process, nitrogen cycle metabolic process) were evaluated

based on UniProt and their fold change. Contig assemblies that were consistently upregulated in

the treated T (relative to treated S and non-treated T) with a significant P-value in the DESeq

analysis were selected, for a total of 49 contigs. A subset of this list was generated based on

known gene function. Contigs with predicted annotations related to stress response, signaling,

transcription factors, and herbicide metabolism were selected as potential candidate NTS genes

involved in glufosinate tolerance.

Candidate gene validation by qRT-PCR

Two A. palmeri populations were treated with glufosinate at 0.37 kg ha-1 using the previously

described procedure. Leaf tissues were collected three days before and 24 h after herbicide

treatment. Tolerant and susceptible plants from each population were identified three weeks after

herbicide treatment based on level of injury. Three biological replicates from the non-treated and

treated samples from each biotype within each population were used for the validation

experiment. Total RNA was extracted from leaf tissues using PureLink RNA Mini kit (Life

Technologies, Carlsbad, CA). Genomic DNA was removed using DNAse I (Thermo Scientific,

Waltham, MA). cDNA was generated from 5 µg total RNA using Reverse Transcription System

first-strand cDNA synthesis kit ( Promega). A 4-fold serial dilution of cDNA samples (1:1, 1:5,

1:25, 1:125) was used to construct a standard curve. Seven of the candidate NTS genes were

46

subjected to real-time quantitative PCR with primers designed using Primer3 tool. Two genes (β-

tubulin, RNA helicase) were used as internal controls for normalization of gene expression.

Primers had an amplification efficiency of 96 to 110%.

The expression level of 7 candidate NTS genes was measured in 24 plants. Quantitative real-

time PCR reactions were conducted in a 12-µL volume containing 6.25 µL of SyberGreen Master

Mix, 1 µL of 1:25 diluted cDNA, and 0.5 µL of 10 µM primers (1:1 mix of forward and reverse

primers). Amplification was performed in a Bio-Rad MiniOpticon System PCR machine (CFX96,

Bio-Rad Laboratories, Inc., Hercules, CA, USA) under the following conditions: 10 min at 94 oC,

40 cycles of 94 oC for 15 s and 60 oC for 1 min, followed by a melt-curve analysis to confirm

single PCR product amplification. Data were analyzed using CFX Manager software (v.1.5).

Slopes for target and internal control genes were equivalent as observed in amplification plots.

Comparative CT method was used to calculate relative expression levels as 2−ΔΔCt where ΔCT =

[CT target gene–geometric mean (CT internal control genes)] and ΔΔCT = [ΔCt tolerant - ΔCt

susceptible]. Wilcoxon non-parametric test (α=0.05) was used to determine statistical difference

in gene expression between tolerant and susceptible biotypes.

Results

Differential response of A. palmeri accessions to glufosinate

The majority of accessions were 100% sensitive to 0.55 kg ha-1 glufosinate, except for some,

which had few survivors. The 120 accessions differentiated into three groups based on mortality

and levels of injury of survivors (Table 1). The first group is composed of the 88 most sensitive

accessions. The second group, composed of 28 accessions, were controlled 94 to 99% with

survivors incurring 60-99% injury. The third group consisted of four apparently segregating

47

accessions with 88 to 97% mortality. Survivors from these accessions incurred 30-80% injury.

Of the possibly segregating accessions, only survivors from 08-Lee-C were able to produce

sufficient seeds to continue further experiments and evaluation. Seven percent of 08-Lee-C

survived glufosinate treatment, of which 4% of treated plants had <61% injury and produced

seeds. Survivors from other recalcitrant accessions were not able to produce enough seeds due to

either having high injury (>75%) or low number of survivors, which were all males. Considering

that plants growing in the field tend to be more robust than those in the greenhouse, the

likelihood of expected escapes in the field may be higher than that in the greenhouse. Plants in

the field also do not receive uniform amounts of herbicide for various reasons such as partial

coverage by other plants or differential plant size. In addition, plants maintained in the

greenhouse that are well-watered and cultured under warm temperatures grow faster and reach

the recommended spraying heights earlier than those growing in the field due to less variation in

ambient conditions in the greenhouse [60].

Response of 08-Lee-C to other foliar herbicides

The 08-Lee-C accession was susceptible to dicamba and fomesafen, but resistant to

glyphosate and ALS inhibitors, which are commonly used herbicides. The mortality of 08-Lee-C

was 98% and 99% with fomesafen and dicamba, respectively (Table 2). On the other hand, 08-

Lee-C was controlled poorly with glyphosate (EPSPS inhibitor) (61%) as well as with ALS

inhibitors trifloxysulfuron (25%), and pyrithiobac (21%). This accession is resistant to two

commonly used modes of action, thus limiting the herbicide options for post-emergence weed

mitigation.

48

Resistance level to glufosinate

The response of A. palmeri to 11 doses of glufosinate fitted a sigmoidal, logistic function

(Fig. 1). The glufosinate doses required to reduce growth by 50% (GR50) were 0.076, 0.110, and

0.214 kg ha-1 for SS, 08-Lee-C, and C1 accessions, respectively (Table 3). Based on these GR50

values, the level of tolerance to glufosinate in 08-Lee-C and and C1 accessions was 1.4- and 2.8-

fold, respectively, relative to the susceptible standard (SS). The GR50 increased 2-fold, from 110

g ha-1 in 08-Lee-C to 214 g glufosinate ha-1 in C1.

Ammonia accumulation in response to glufosinate

Glutamine synthetase, the target site of glufosinate, is a nuclear-coded enzyme that catalyzes

the conversion of L-glutamate to L-glutamine by assimilating ammonia in the cytoplasm and

plastids, but predominantly in the chloroplast of green tissues [21]. Ammonia accumulation is a

direct response to the inhibition of this pathway by glufosinate. The ammonia concentration in S

plants was 830 µg g-1 fresh leaf tissue (+60) and was 394 µg g-1 fresh weight (+40) in T plants

(Fig. 2). The S plants accumulated 2X more ammonia than the T plants, indicating rapid

depletion of functional glutamine synthetase as a consequence of glufosinate treatment.

Glutamine synthetase 2 (GS2) relative copy number

The relative GS2 copy number of S and T plants ranged from 1 to 3 (Fig. 3). Similar GS2

copies were detected in both S and T plants, indicating that differential tolerance to glufosinate is

not due to amplification of the GS2 gene. Transcriptome analysis also revealed that GS2 was not

49

differentially expressed between T and S plants, which indicated that differential tolerance to

glufosinate in A. palmeri was not due to changes in expression of the target enzyme.

Glutamine synthetase gene sequence analysis

Glutamine synthetase plays a primary role in plant nitrogen metabolism by catalyzing the

conversion of glutamate to glutamine [21]. Glutamine synthetase in higher plants exists in two

major isoforms: GS1 in the cytosol and GS2 in the chloroplast/plastids [61]. The cytosolic form

(GS1) is the predominant isoform in roots and non-green tissues [62]. The chloroplast form of

glutamine synthetase (GS2) is the major isoform in leaves, which is primarily responsible for

recycling ammonia during photorespiration and synthesis of glutamine [63]. In our study, two

different alleles of GS2 were observed in the T plants and one in the S plants. The nucleotide

sequences obtained from the two biotypes had 97-99% identity with GS2 sequences from other

Amaranthus species (A. albus, A. hybridus, A. spinosus, A. lividus, A. thunbergii, and A. viridis).

The T biotype differed in six amino acids in the upstream region of GS2 when compared to the S

biotype. Seven nonsynonymous point mutations (Tyr8Asn, Ser25Leu, Asn26Ser, Lys37Gln,

Gly39Lys, Gln54Lys, Asp56Glu) in the upstream region were detected in one of the GS2 alleles of

the T biotype (Fig. 4). The second allele of the T biotype harbored only the Tyr8Asn substitution.

These nonsynonymous substitutions identified in the A. palmeri T biotype also occur in

herbicide-susceptible A. viridis, indicating that these substitutions do not contribute to tolerance

to glufosinate. Other nucleotide polymorphisms between T and S plants were synonymous

mutations.

50

Global transcriptional changes in A. palmeri 24 h after glufosinate application

A reference cDNA transcriptome consisting of 72,794 sequences was assembled (Table 4).

Treatment samples were similar as indicated in the multidimensional scaling (MDS) plot (Fig.

5). Biological replicates from the same treatment clustered together indicating low bias and

variation among treatment samples. One or more GO terms were assigned to 33,516 sequences

with 76,455 GO assignments in total for biological process (31.9%), cellular component (10.8%)

and molecular function (57.3%) categories.

Background differences between susceptible and tolerant plants. Pairwise comparison

between non-treated S and T plants showed 438 differentially expressed genes, 158 of which

were downregulated and 280 were upregulated in the T plants relative to S plants (Table 5).

Genes that were notably more expressed in the T plants relative to the S plants without herbicide

treatment included cytochrome P450s (Cyp72A219, Cyp86b, Cyt77A, Cyt71A, Cyt76A, Cyt86A),

transporters (ABC transporter), transferases (glycosyltransferase, acyltransferases), antioxidants

(glutathione-S-transferase, superoxide dismutase), and genes related to lipid metabolism

(esterase lipase).

Genes induced by glufosinate application. Relative to the respective non-treated checks,

8154 genes were affected by glufosinate application in the T plants and 6034 genes in the S

plants (Table 5, Figs. 6 and 7). Comparison between treated T and S plants revealed 567 genes

that were more repressed or more induced by glufosinate in the treated T plants. Overall, there

were 210 upregulated and 357 downregulated genes in the treated T relative to the treated S

plants (Figs. 8 and 9). One hundred-five glufosinate-responsive genes were differentially

expressed in both treated T (32 genes) and S plants (73 genes) (Fig. 7). In addition, 239 genes

that were differentially expressed between treated (52 genes) and non-treated (187 genes) T

51

plants were more highly repressed or more induced in treated T plants than in treated S plants. Of

these 239 genes, the majority were related to biosynthetic process, cellular nitrogen compound

and small molecule metabolic processes, response to stress, and oxidoreductase activity (Fig. 9).

Among the upregulated genes of this 239-gene subset, 91 were induced by glufosinate in T

plants, including genes putatively annotated as NAC transcription factor, CYP94a1, and ABC

transporter b. The majority of upregulated genes that were differentially expressed between

treated T and S plants, and that were also differentially expressed relative to their respective non-

treated counterparts, were related to nitrogen compound metabolic processes, oxidoreductase

activity, nucleotide binding, and transferase activity. Two genes that were repressed exclusively

in the treated T plants relative to the treated S plants were folypolyglutamate synthase and

caffeic acid 3-o-methyltransferase (Fig. 7).

The functional classification of selected differentially expressed genes (DEGs) associated

with stress response and herbicide metabolism was examined to investigate the pattern of

transcriptome regulation that occurred during glufosinate treatment (Fig. 11). Glufosinate

treatment triggered the expression of genes related to stress response and xenobiotic

detoxification as expected. Some genes associated with photosynthesis, structural stabilization,

cell membrane binding, stress response, and detoxification were repressed. Increased expression

of non-target site (NTS) genes related to stress response, stress signaling, detoxification, abiotic

response, cell structure stabilization, and growth and senescence was observed in treated T

plants. Genes that were exclusively induced in treated T plants were annotated to code for

transmembrane protein 45b, heat stress transcription factor B, hypersensitive-induced response

protein, cytochrome P450 (Cyp72A219, 94A2, Cyt86b1-like), transcription factor, ethylene-

responsive transcription factor, glutathione S-transferase (GST), zinc finger protein constans-like

52

10, and NAC transcription factor (Fig 8, Tables 5 and 6). These candidate genes likely play a role

in the adaptation of A. palmeri to glufosinate and, possibly, also to other herbicides.

Of the 239 genes that were more highly induced or repressed in the treated T plants, 49

were consistently upregulated. These consistently upregulated genes are involved in biosynthetic

process, cellular nitrogen compound metabolic process, nucleic acid binding transcription factor

activity, oxidoreductase activity, stress response, signal transduction, transferase activity,

transmembrane transporter activity, and transport (Table 6 and Fig. 10). A subset of 13

glufosinate-inducible genes which are related to detoxification, stress signaling, and transport are

candidate genes involved in conferring some tolerance to glufosinate. These include ABA 8’-

hydroxylase, ABC transporter, chitin elicitor receptor kinase, cytochrome P450 72A, cytochrome

P450 94A, GST, heat stress transcription factor, heat shock protein 83, ethylene response

transcription factor, NAC transcription factor, NAC transcription factor 25, NADH ubiquinone

oxidoreductase, and nitronate monooxygenase (NMO) (Table 7). These genes were induced >2-

fold in treated T plants relative to treated S plants and non-treated T plants.

Validation of selected genes using qRT-PCR

The expression of seven candidate NTSR genes was measured in two A. palmeri accessions

using quantitative real-time PCR. Gene expression was similar in the non-treated tolerant and

susceptible plants. The 7 genes were induced in T and S plants upon glufosinate treatment. Five

genes (HSP, NMO, ETF, ABC, NAC) were not differentially expressed between treated tolerant

and treated susceptible plants. GST was differentially expressed in only one of the treated tolerant

plants. On the other hand, CYP72A219 was expressed eight times higher in all the treated tolerant

plants relative to the susceptible ones (Fig. 12).

53

Discussion

Differential tolerance to glufosinate in A. palmeri accessions

The differential response to glufosinate in the 120 Amaranthus palmeri accessions

demonstrates variation in herbicide efficacy on a weed population (Table 1). Several factors

affect glufosinate activity even on a single species; these include temperature, light, relative

humidity, time of day, plant size/age, and dose [30, 64-69]. Variation in environmental

conditions was minimal in the greenhouse. Variation in plant factors was minimized by

maintaining seedlings of the same size. The impact of time of day on glufosinate activity was

eliminated by applying the herbicide at about the same time in each repetition of the experiment.

Accessions with a few survivors reflect some heterogeneity within the population, as expected of

this highly diverse species. Projecting such diversity to field conditions, where all the factors

mentioned above can vary, we expect to see a higher frequency of individuals that would escape

weed control activities in the field. Many such escapes could have been subjected to sub-lethal

doses as a consequence of plant, environmental, and application variables. Controlling these

escaped individuals, or adopting a weed management strategy that controls escaping genotypes,

is a critical step in mitigating the accumulation of non-target-site genes that could eventually

endow resistance to herbicides [70].

Resistance profiling of the 08-Lee-C recalcitrant accession

Herbicides impose strong abiotic stresses to weeds in crop fields, managed turfgrass,

gardens, and roadsides. The evolution of resistance in populations of weedy species is an

increasing problem worldwide. The recalcitrant A. palmeri accession, 08-Lee-C, was resistant to

54

glyphosate and ALS-inhibiting herbicides, trifloxysulfuron and pyrithiobac (Table 2). The

occurrence of resistance to multiple herbicides in 08-Lee-C is not surprising because this field

was sprayed with glyphosate and ALS inhibitors for several years. This field was planted with

glyphosate-tolerant crops for more than three years and had been exposed to ALS inhibitors in

the years prior when the grower was planting conventional soybean. Resistance to glyphosate

and ALS inhibitors among A. palmeri in Arkansas is widespread [71]. Fomesafen, a PPO

herbicide with soil and foliar activity, was first commercialized in the 1960s and had been used

by farmers mainly for soybean. The usage of fomesafen, and almost all other herbicides in

soybean, dropped when glyphosate-tolerant soybean was introduced in the mid-1990s. Upon the

explosion of glyphosate-resistant Amaranthus species, soybean farmers reverted to using

fomesafen and its use was expanded to cotton to control glyphosate-resistant Amaranthus

species. The farmer of this field, like many others, had been using only glyphosate to control

weeds. Although glufosinate had not been used in this field, some A. palmeri individuals

displayed differential tolerance to glufosinate. Being an obligate outcrossing species, A. palmeri

exhibits high genetic diversity, which facilitates its tendency to evolve herbicide resistance.

Intensive use of glufosinate in this field, in a manner that allows escapes to produce seed, will

accelerate the evolution of resistance through accumulation of multiple low-impact tolerance

genes as demonstrated already in some species, including Lolium rigidum [72] and A. palmeri

[73]. Tolerance traits can accumulate and get fixed in the population as selection pressure

continues.

55

Ammonia accumulation in response to glufosinate

Ammonia accumulation is directly related to glufosinate toxicity. Inhibition of glutamine

synthetase and ammonia accumulation triggers a cascade of reactions, including inhibition of

ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) enzyme [74] and photosystem

electron flow [75], affecting photosynthesis [30, 76] leading to plant death. Ammonia reduces

pH gradient across the membrane, which uncouples photophosphorylation [75]. Elevated levels

of ammonia accumulated in glufosinate-treated rice and soybean cell cultures [77, 78]. In our

study, glufosinate-sensitive plants accumulated 2X more ammonia than the T plants (Fig. 2).

Similarly, glufosinate-sensitive L. perenne ssp. multiflorum from Oregon accumulated 1.6X

more ammonia than the resistant population [25]. Increased ammonia level in S plants after

glusofinate treatment is the consequence of rapid depletion of functional glutamine synthetase.

Reduced ammonia accumulation in T plants indicates the presence of mechanism(s) that reduce

the impact of glufosinate on plant function. Such mechanisms could either be reduced binding

affinity of glufosinate by target site modification or, mechanisms external to the herbicide-

binding site (NTSM) including detoxification and others. The latter applies to the glufosinate-

tolerant A. palmeri plants.

Glutamine synthetase (GS2) copy number

Gene amplification conferring herbicide resistance has been identified in GR weeds such as

A. palmeri, A. tuberculatus, K. scoparia, and L. multiflorum [43, 79, 80]. The GR plants contain

multiple copies of EPSPS, the target site of glyphosate, which results in increased production of

EPSPS enzyme allowing the plant to function normally despite the presence of glyphosate. This

mechanism has not been observed with other herbicide target genes either because it is exclusive

56

to the EPSPS regulatory process, or simply because it has not been investigated in other

herbicide target genes. Amplification of GS2, in glufosinate-resistant weeds, is not yet reported.

However, a 4- to 11-fold amplification of GS2 in alfalfa cell culture lines resulted in increased

GS enzyme production, endowing resistance to glufosinate [81]. In our study, S and T A. palmeri

had similar copies (1-3) of GS2 indicating that tolerance to glufosinate was not due to GS2

amplification (Fig. 3). This was supported by the fact that GS2 transcripts were not different

between S and T plants.

Glutamine synthetase (GS2) gene sequence

A rare individual in a population may harbor a mutation in the herbicide-binding site that can

alter the folding structure of the protein, resulting in reduced binding affinity of the herbicide.

Resistance to ALS-inhibiting herbicides in weeds, in most cases, is due to mutation(s) in one or

more of the binding domains in the ALS enzyme [82]. The higher frequency of SNPs in GS2 of

T plants could predispose such individuals in the population to accumulate nonsynonymous

nucleotide substitutions. However, genetic polymorphisms may not always get translated to

protein polymorphisms, and only certain amino acid mutations will result in herbicide resistance

[82]. In the current study, most of the polymorphisms observed in the nucleotide sequence of

GS2 in T plants were synonymous. Although seven amino acid substitutions in the upstream

region were detected in one of the alleles of the T biotype, these substitutions also occur in

glufosinate-sensitive A. viridis and is in a region outside of the substrate-binding domain (Fig. 4).

A Tyr8Asn substitution was detected in the two GS2 alleles of the T biotype. Asparagine and

tyrosine are both polar, uncharged amino acids, hence Tyr8Asn substitution may not alter the

physiological and physicochemical stability of the plastid GS enzyme. The presence of this

57

trivial amino acid substitution in glufosinate-tolerant plants as well as similar GS2 copies as that

of the S plants suggests that non-target-site tolerance factors are involved. Transcriptome

analysis could inform us on differential tolerance mechanisms. A glufosinate-resistant L. perenne

ssp. multiflorum from Oregon, USA, which showed similar level of ammonia accumulation to

the tolerant A. palmeri in our study, harbors a Asn171Asp mutation (GAC TO AAC) in the GS2

gene which confers resistance to glufosinate [25].

Tolerance level to glufosinate in 08-Lee-C and C1

After one cycle of selection, the GR50 for C1 increased from 1.44-fold to 2.80-fold relative to

SS, reflecting increased frequency of tolerant plants in C1 (Table 3). Although a low frequency

(<10%) of the plants survived exposure to glufosinate, the increase in GR50 after one cycle of

glufosinate selection is indicative that the population has become less sensitive to glufosinate

after one cycle of purifying selection. Because glufosinate had not been used in the field, the

frequency of survivors was low, and the tolerance level was only 1.44 to 2.80-fold, it is likely

that these plant variants have low-level, non-target site resistance. We are possibly capturing an

early phase of herbicide resistance evolution. Should the population continue to be under

selection pressure from glufosinate, the probability of the population acquiring additional

adaptive alleles and expressing resistance to field use rate of glufosinate would increase.

Transcriptome of A. palmeri and candidate NTS genes involved in glufosinate tolerance

A reference cDNA transcriptome consisting of 72,794 sequences was assembled for A.

palmeri (BioProject PRJNA390774, TaxId 107608). The transcriptome of A. hypochondriacus

58

had 57,658 assembled sequences [36]. Our data demonstrated broad effects of glufosinate on

several metabolic pathways, as expected of a herbicidal compound, including nitrogen

assimilation and metabolism similar to what is reported in Arabidopsis [83]. One of the apparent

functional categories to which glufosinate-responsive genes belong is protein families known to

participate in metabolism, stress response, and defense, with the majority of these genes

potentially associated with abiotic stress response signaling and chemical detoxification

pathways (Figs. 10 and 11). Stress response genes are inducible by many other herbicides or

stress factors. Generally, abiotic stress such as herbicide, salinity, and drought modulates the

expression of genes that are involved in signaling cascades and in transcriptional control [84],

genes that code for proteins involved in membrane protection [85], and genes that are involved in

water and ion uptake and transport [86, 87]. These stress-regulated genes are activated to

counteract the stress effects, maintain homeostasis, and adapt. Cell membrane receptor-kinases,

stress signaling genes, detoxification-related genes, and antioxidants were activated upon

glufosinate treatment in both S and T plants (Fig. 11). Peroxidase and superoxide dismutase, for

example, were upregulated to help counteract the oxidative stress caused by lipid peroxidation

resulting from glufosinate treatment. This indicates that plants undergo extensive transcriptional

adjustment in response to herbicide-induced stress. Activation of herbicide-stress-response genes

is hypothesized to be initiated by a herbicide sensor, which triggers the activation of regulator

genes, which causes a cascade of reactions to either detoxify the herbicide or protect the plant

from herbicide-mediated stress [35].

Two genes, folypolyglutamate synthase and caffeic acid 3-o-methyltransferase, were

repressed in the treated T relative to S plants, but were not differentially expressed in other

pairwise comparisons. Both are involved in one-carbon transfer and phenylpropanoid

59

biosynthesis [88, 89]. The phenylpropanoid pathway serves as a rich source of metabolites in

plants, especially for lignin biosynthesis and the production of flavonoids, coumarins,

hydrocinnamic acid conjugate, cutins and lignins [90, 91]. Phenylpropanoids are involved in

plant defense, structural support and survival [91, 92]. Repression of genes involved in

phenylpropanoid biosynthesis indicate less allocation of carbon resources to these plant products

in the T plants after glufosinate treatment compared to the S plants, indicating carbon allocation

to other intermediates is more critical for survival under herbicide stress.

Thirteen candidate genes were identified which included ABA 8’-hydroxylase, ABC

transporter (ABC), chitin elicitor receptor kinase (CERK1), cytochrome P450 72A

(CYP72A219), cytochrome P450 94A, glutathione S-transferase (GST), heat stress transcription

factor, NAC transcription factor, NAC transcriptor 25, ethylene-response transcription factor

(ETF), heat shock protein 83 (HSP), NADH ubiquinone oxidoreductase and nitronate

monooxygenase (NMO) (Table 6). The expression of these candidate genes was induced by

glufosinate treatment. Delye [35] proposed a model of NTS resistance mechanism in which

herbicide stress triggers the expression of ‘protectors’ and ‘regulators’, as well as epigenetic

modifiers which enable the plant to survive herbicide stress. Protector genes include cytochrome

P450, oxidase, peroxidases, esterases, hydrolases, glutathione S-transferases and transporters,

which play roles in reducing the efficacy of herbicide by detoxification. ‘Regulator’ genes are

involved in transcriptional, post-transcriptional, and post-transductional control such as

transcription factors, micro-RNAs, and kinases [35]. It is noteworthy that the candidate NTS

genes could act as either ‘protector’ or ‘regulator’ based on their functions. Cytochrome P450,

GST, NADH ubiquinone oxidoreductase, and ABC transporter proteins have roles in pesticide

detoxification [93-98]. Chitin elicitor receptor kinase, ABA 8’-hydroxylase, ethylene-response

60

transcription factor, heat stress transcription factor, and NAC transcription factor are involved in

stress response signaling and regulation [35, 89, 99-102].

Of the seven candidate genes subjected to qRT-PCR validation experiment, only two genes,

(cytochrome P450 CYP72A219 and GST) were associated with tolerance to glufosinate (Fig. 12).

The induction of GST and CYP72A219 suggests that T plants are able to deactivate glufosinate to

some extent. Induction of cytochrome P450 and GST facilitate the conversion of the herbicide

into a less toxic metabolite. The biochemical role of cytochrome P450-mediated herbicide

metabolism has been well established in herbicide-resistant weed species. Plant cytochrome

P450s facilitate the detoxification of toxic xenobiotics by catalyzing oxygen- and NADPH-

dependent mono-oxygenation reactions which convert herbicide into a more hydrophilic

metabolite [103]. Hundreds of P450 genes exist in higher plants. For example, Arabidopsis

thaliana and Oryza sativa possess 272 and 458 putative P450 genes, respectively [104]. RNA-

Seq transcriptome analysis of L. rigidum identified CYP72A genes to be involved in metabolic

resistance to diclofop [37]. Non-target-site ACCase and ALS resistance in Alopecurus

myosuroides [95, 96], Stellaria media [105], Lolium [96, 106-108], Sinapis arvensis [109],

Echinochloa phyllopogon [110], and Digitaria sanguinalis [111] were reported previously to be

facilitated by cytochrome P450 enzymes. Upregulation of CYP72A and CYP94A was reported in

a multiple-herbicide-resistant E. phyllopogon population [112]. Similarly, CYP94A1, a plant

cytochrome P450-catalyzing fatty acid omega hydroxylase, was induced by chemical stress in

Vicia sativa and by bentazon treatment in soybean [113, 114]. Some cytochrome P450 genes in

the CYP71A family were also demonstrated to be involved directly in herbicide metabolism in

crops, such as O. sativa CYP71A31 and Zea mays CYP71A28 [115, 116].

61

The involvement of glutathione S-transferases (GSTs) in herbicide resistance is reported in

several weed species. Glutathione S-transferases are ubiquitous enzymes that catalyze the

conjugation of harmful xenobiotics to reduced glutathione, facilitating their metabolism,

sequestration or removal [117]. The primary factor for atrazine selectivity in corn is the activity

of a soluble GST, which detoxifies atrazine by forming an atrazine-glutathione conjugate [118].

In a recent transcriptome study, increased expression of GST is associated with diclofop

resistance in L. rigidum and nicosulfuron tolerance in Z. mays [37, 116]. GST also functions as

an antioxidant, protecting plants from herbicide-mediated oxidative stress by scavenging reactive

oxygen species [119]. Increased expression of glutathione transferase gene (AmGSTF1) in

multiple-resistant A. myosuroides led to accumulation of flavonoids which protects the plant

from herbicide injury [120]. It has been reported that glutathione transferase orchestrate

tolerance to abiotic stress through their ability to regulate redox signaling pathways that activate

defense genes [121]. In glufosinate-tolerant A. palmeri, GST is possibly involved in converting

glufosinate into a less toxic metabolite following possible minimal phase I detoxification by

CYP72A219 as well as in protecting the plants against oxidative stress and lipid peroxidation

from glufosinate phytoxicity.

Other candidate genes were identified by RNASeq, but were not differentially expressed in

the validation experiment, including as the ABC transporter, NAC transcription factor, NMO,

HSP, and ethylene transcription factor. Although these might not be involved in conferring some

level of tolerance to glufosinate, their involvement in detoxification of toxic xenobiotic

compounds and in stress response have been reported. For example, NMO in A. thaliana is

associated with detoxification of the allelochemical benzoxazolin [122]. Increased expression of

NADH ubiquinone oxidoreductase and induction of P450 genes were involved in resistance to

62

pyriproxyfen insecticide in Bemisia tabaci [123]. Induction of heat shock proteins is associated

with drought and oxidative stress in Brassica juncea [124] and A. thaliana [125], respectively.

Plant ABC transporters have been associated with the movement of herbicide conjugates [126,

127]. Modifications of ABC transporters have been suspected in some cases of weed resistance

to glyphosate or paraquat [128, 129]. Tolerance of Arabidopsis thaliana to paraquat is endowed

by a mutation in the plasma membrane-localized ABC transporter, which resulted in reduced

herbicide uptake in plant cells [130]. NAC transcription factors and ethylene-response

transcription factors play an important role in the regulation of the transcriptional reprogramming

associated with plant stress response such as cold, drought, and salinity [131-134].

Diversity in gene expression and regulation is an important factor driving herbicide

resistance evolution [135]. As gene expression regulation also involves post-transcriptional and

post-translational controls, protein expression of the identified genes may need to be further

investigated. Because of genetic diversity, plants have the potential to overcome herbicide stress

through a concerted effort of multiple genes. Plants with low-level tolerance showed greater

induction of abiotic stress-protection- and detoxification-related genes than S plants. Thus,

survival from glufosinate treatment is facilitated by stress-protection/stress-adaptation genes.

Differential expression of stress-protection genes in a population can enable some individuals to

survive herbicide application. Tolerance-related genes can get fixed in a population upon the

exertion of sustained selection pressure. Selection pressure coupled with genetic diversity drives

evolutionary processes leading to herbicide resistance.

A low frequency of a low-level presumed tolerance in the phenotype was observed across a

two-stage screening of survivors from a recalcitrant, segregating population. The surviving

individuals were described as tolerant because there was no record of their exposure to

63

glufosinate in this field. However, a three-year record, which is what growers can provide

generally, is insufficient to assert that the gene regulation demonstrated here is truly ancestral

and that it would have been expressed in the total absence of glufosinate selection. Amaranthus

palmeri is a prolific dioecious plant. Seed could have been transported to the field from

elsewhere where glufosinate has been used. Similarly, pollen from other fields with a history of

glufosinate use, could have blown through the field and fertilized female plants. Differential

gene expression of CYP72A219 and GST and the presence of surviving progeny in intentionally

intercrossed populations show that genetic mechanisms exist for the evolution of low-level, or

potentially incipient, resistance to glufosinate in some A. palmeri populations.

Natural variation in sensitivity to herbicides among individuals exists in A. palmeri

population. The inherent ability of A. palmeri to tolerate glufosinate, or any other herbicides, due

to non-target mechanism(s) will impact the dynamics and evolution of herbicide-resistant

populations. The frequency of glufosinate-tolerant biotypes in a population will increase with

continued herbicide exposure. Non-target-genes could accumulate and get fixed in the population

leading to evolution of resistance. Vigilance will be required to detect elevated glufosinate

tolerance, especially because it is multi-genic, impossible to eliminate from populations, and

eventually might confer resistance to more than one class of herbicide chemistries and abiotic

stresses. If proactive measures are ignored, it is just a matter of time that glufosinate-resistant A.

palmeri populations will evolve and cause formidable task in weed management.

64

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New Phytol. 2009;184:783-93. doi: 10.1111/j.1469-8137.2009.03034.x

74

Table 1. Cluster analysis of A. palmeri accessions treated with glufosinate at 0.55 kg ha-1.

Cluster Number of

accessions

Mortality (%) Mean frequency of plants at different levels of injury (%)

Mean Min Max 0-10%

injury

11-30%

injury

31-60%

injury

61-80%

injury

81-99%

injury

100%

injury

1 88 100 100 100 0 0 0 0 0 100

2 28 98 94 99 0 0 1 1 0 98

3 4 92 88 97 0 1 3 4 0 92

75

Table 2. Response of A. palmeri (08-Lee-C) to foliar-applied herbicides.

Herbicide Mortality (%)a Mode of actionb

Dicamba 99 Synthetic auxin

Fomesafen 98 PPO inhibitor

Glufosinate 93 Glutamine synthetase inhibitor

Glyphosate 61 EPSP synthase inhibitor

Pyrithiobac 21 ALS inhibitor

Trifloxysulfuron 25 ALS inhibitor

aUniform-sized plants (7.5-9 cm tall) were sprayed with dicamba (280 g ha-1), fomesafen (264 g

ha-1) glufosinate (0.55 kg ha-1), glyphosate (870 g ha-1), pyrithiobac (73 g ha-1), and

trifloxysulfuron (8 g ha-1). Mortality was recorded 21 d after herbicide application. bPPO- protoporphyrinogen oxidase, EPSP- enolpyruvyl shikimate-3-phosphate, ALS- acetolactate synthase

76

Table 3. Glufosinate dose required to reduce growth by 50% (GR50) in A. palmeri 08-Lee-C, C1

and SS accessions.

Accession GR50

Confidence

Intervalsa

T/Sb

kg ai ha-1

08-Lee-C 0.110

0.097 - 0.123 1.44

C1 0.214

0.184 - 0.244 2.80

SSc 0.076

0.064 - 0.088

a 95% confidence intervals.

b Tolerance levels (T/S) calculated using the GR50

of the tolerant accession relative to the susceptible standard. cHerbicide-susceptible standard accession.

77

Table 4. Summary of statistics for transcriptome assembly.

Reads (n) Bases (Mb) Average

length (bp)

Illumina raw reads 1,667,277, 670 8409.7 125

Assembled contigs 72,780 49.15 675

Annotated sequences (blastX) 65,282 - -

Sequences assigned with GO terms 33,294 - -

78

Table 5. Differentially expressed genes putatively involved in differential tolerance to glufosinate in Amaranthus palmeri.

Level of

gene

expression

Number of differentially expressed genes

TWO vs SWO SWT vs SWO TWT vs TWO TWT vs SWT

Repressed Induced Repressed Induced Repressed Induced Repressed Induced

>1-2 2 0 589 353 1277 1048 0 0

>2-3 43 26 1228 994 1846 1229 33 11

>3-4 58 72 662 677 756 679 94 65

>4-5 27 64 328 389 299 351 74 41

>5-6 13 36 109 218 125 182 63 31

>6-7 4 22 72 146 55 135 23 22

>7-10 8 51 45 175 37 113 47 33

>10 3 9 10 39 1 21 23 7

Total 158 280 3043 2991 4396 3758 357 210

438 6034 8254 567

aTWO vs SWO: non-treated tolerant (T) relative to non-treated susceptible (S) plants; SWT vs SWO: treated S relative to non-treated S plants;

TWT vs TWO: treated T relative to non-treated T plants; TWT vs SWT: treated T relative to treated S plants

79

Table 6. Upregulated genes in glufosinate-treated and non-treated tolerant (T) plants, and in

glufosinate-treated T relative to treated susceptible (S) plants, assigned with Gene Ontology

molecular function and biological process related to metabolism and signaling pathways.

GO function Contig Gene annotation Fold change

TWTn/TWOn TWTn/SWTn

biosynthetic process Pa27529 50S ribosomal protein

chloroplastic 8.47 9.02

biosynthetic process Pa29824 60s ribosomal protein l13a-

2 7.60 6.25

biosynthetic process Pa38623 phenazine biosynthesis-

like domain-containing

protein 1 isoform x2

6.93 3.92

biosynthetic process, cellular

nitrogen compound metabolic

process, nucleic acid binding

transcription factor activity

Pa65724 cyclic dof factor 1-like

3.67 4.46

biosynthetic process, small molecule

metabolic process

Pa35601 phosphoribosylaminoimida

zole chloroplastic-like 9.24 11.22

cellular amino acid metabolic

process, biosynthetic process, small

molecule metabolic process

Pa17844 shikimate chloroplastic

2.49 4.41


process, secondary metabolic process

Pa39917 3-isopropylmalate

dehydratase -like protein 4.86 5.79


process, small molecule metabolic

process

Pa60555 probable low-specificity l-

threonine aldolase 1 7.04 5.40

cellular nitrogen compound

metabolic process

Pa52820 putative polyprotein 2.55 4.88


metabolic process

Pa63676 CTP synthase 1.64 3.18


metabolic process

Pa71553 gag-pol polyprotein 5.93 7.09


metabolic process

Pa8879 zinc finger bed domain-

containing protein

ricesleeper 1-like

7.63 9.05


metabolic process, biosynthetic

process, signal transduction

Pa63868 two-component response

regulator arr9 isoform x1 2.20 3.43



process

Pa37812 NAC transcription factor

8.25 7.80




Pa51700 two-component response

regulator arr5-like isoform

x2

1.62 2.58


metabolic process, response to stress,

biosynthetic process

Pa37809 heat stress transcription

factor b-2b-like 3.01 2.88


metabolic process, response to stress,

biosynthetic process, signal

transduction

Pa13900 RNA polymerase ii c-

terminal domain

phosphatase-like 1 3.52 3.83

hydrolase activity, acting on glycosyl

bonds

Pa26833 PREDICTED: alpha-

glucosidase-like [Beta

vulgaris subsp. vulgaris]

6.20 4.47

80

Table 6 (Cont.)


TWTn/TWOn TWTn/SWTn


bonds

Pa42036 alkaline neutral invertase

cinv2-like 3.40 3.15


bonds, response to stress

Pa69030 beta-amylase chloroplastic 3.94 5.41

nucleic acid binding transcription

factor activity, biosynthetic process,

signal transduction

Pa49594 auxin-responsive protein

iaa29 4.11 4.51


factor activity, cellular nitrogen

compound metabolic process,

biosynthetic process

Pa47424 NAC transcription factor 25-

like 1.98 3.34


factor activity, cellular nitrogen

compound metabolic process,

response to stress, biosynthetic


Pa38292 ethylene-responsive

transcription factor abr1

5.24 4.92

oxidoreductase activity Pa10467 cytochrome P450

cyp72A219-like 8.56 4.42

oxidoreductase activity Pa40402 internal alternative NAD H-

ubiquinone oxidoreductase

mitochondrial

5.22 3.49

oxidoreductase activity Pa45867 nitronate monooxygenase 6.26 4.28

oxidoreductase activity Pa51578 -dopa dioxygenase extradiol-

like protein 6.44 5.06

oxidoreductase activity Pa56011 short-chain type

dehydrogenase reductase-like 7.89 3.99

oxidoreductase activity Pa60473 cytochrome P450 94a1-like 2.90 3.34

oxidoreductase activity, cellular

amino acid & metabolic process,


process, homeostatic process,

oxidoreductase activity

Pa10326 5 -adenylylsulfate reductase

chloroplastic- partial

3.47 4.04

oxidoreductase activity, small


Pa44392 abscisic acid 8 -hydroxylase

2

4.153201 4.072155

response to stress, immune system

response

Pa52955 macpf domain-containing

protein at1g14780

1.801976 3.089814

response to stress, immune system

response

Pa62900 heat shock protein 83 4.420622 4.392136

response to stress, signal

transduction, immune system process

Pa42133 chitin elicitor receptor kinase

1-like

3.354966 3.353194

response to stress, signal

transduction, immune system process

Pa69811 receptor-like protein kinase

at3g47110

3.401606 6.459389

response to stress, transport,

transmembrane transport

Pa53135 mitochondrial phosphate

carrier protein mitochondrial-

like

11.42314 4.210532

signal transduction Pa60381 receptor-like serine

threonine-protein kinase sd1-

8 isoform x1

5.532361 3.68854

signal transduction Pa63442 PREDICTED:

uncharacterized protein

LOC104887975

3.319583 5.558233

81

Table 6. (Cont.)


TWTn/TWOn TWTn/SWTn

transferase activity, transferring acyl

groups

Pa67068 uncharacterized

acetyltransferase at3g50280-

like

10.27052 4.376485

transferase activity, transferring alkyl

or aryl (other than methyl) groups

Pa19271 glutathione s-transferase-like

protein

10.47473 6.70153

transferase activity, transferring

glycosyl groups, biosynthetic process

Pa49933 7-deoxyloganetin

glucosyltransferase-like

6.037006 4.239084

transferase activity, transferring

glycosyl groups, response to stress,

bioysnthetic process, small molecule

metabolic process, cell wall

organization or biogenesis

Pa57353 gdp-l-galactose

phosphorylase 2-like

5.457352 5.232313

transmembrane transporter activity Pa14919 peroxisomal nicotinamide

adenine dinucleotide carrier-

like

1.675916 4.465376

transmembrane transporter activity Pa21499 calcium-transporting atpase

plasma membrane-type

4.459466 3.358566

transmembrane transporter activity Pa35784 mate efflux family protein 9-

like

3.45508 3.260395

transmembrane transporter activity Pa63432 anoctamin-like protein

at1g73020

3.788903 3.221265

transmembrane transporter activity,


metabolic process, transport, small


Pa63215 ABC transporter b family

member 2-like

4.662064 6.567808

transport Pa60553 outer envelope protein

mitochondrial

3.146434 7.2662

aTWT = treated T plants

TWO = non-treated T plants

SWT = treated S plants

82

Table 7. Candidate non-target genes, identified by RNA-Seq analysis, that are potentially

involved in conferring differential tolerance to glufosinate in A. palmeri.

Contig Gene annotation Fold changea Function

TWTn/TWOn TWTn/SWTn

19271 Glutathione S-transferase (GST) 10.47 6.7 Detoxification

10467 Cytochrome P450 CYP72A219 8.55 4.42 Heme-thiolate

monoxygenase;

detoxification

37812 NAC transcription factor 8.24 7.8 Transcription

regulator in plant

stress response

38292 Ethylene-response transcription

factor abr1

5.24 4.91 ABA signaling

pathway in

response to stress

response

40402 NAD H-ubiquinone

oxidoreductase

5.21 3.49 Detoxification

63215 ABC transporter b family

member 2

4.66 6.56 Transmembrane

transport

62900 Heat shock protein 83 4.31 8.2 Molecular

chaperone; stress

signaling

44392 ABA 8’-hydroxylase 4.15 4.07 ABA catabolism

45867 Nitronate monooxygenase (NMO) 6.26 4.27 Detoxification

42133 Chitin elicitor receptor kinase 1

(CERK1)

3.35 3.35 Cell surface

receptor toward

biotic and abiotic

stresses

37809 Heat stress transcription factor b 3.01 2.87 Transcription

regulator for heat

shock proteins;

stress signaling

60473 Cytochrome P450 94a1 2.9 3.33 Detoxification

47424 NAC transcription factor 25-like 1.97 3.34 Abiotic stress

response aTWT = treated tolerant plants

TWO = non-treated tolerant plants

SWT = treated sensitive plants

83

Figure 1. Shoot biomass reduction (%) of 08-Lee-C, C1, and SS A. palmeri accessions, 21 days

after glufosinate treatment. Data were best described with nonlinear, sigmoidal, four-parameter

logistic regression function.

84

Figure 2. Ammonia content in glufosinate-tolerant (T) and –susceptible (S) A. palmeri.Error bars

represent standard error. White bars = S plants; gray bars = T plants.

85

Figure 3. Relative copy number of A. palmeri GS2 in glufosinate-susceptible (S) and -tolerant

(T) plants. Error bars represent standard deviation of the mean. Gray bars = T plants; black bars

= T plant.

86

Figure 4. Multiple alignment of the plastidic glutamine synthetase (GS2) amino acid sequences

in Amaranthus. A. palmeri (reference A. palmeri), T1 and T2 = GS2 alleles of glufosinate-

tolerant A. palmeri biotype, S = GS2 sequence of glufosinate-susceptible A. palmeri biotype.

87

Figure 5. Multidimensional scaling (MDS) plot showing the relationship between sample

types.TWO = non-treated tolerant, TWT= treated tolerant, SWO = non-treated susceptible, SWT

= treated susceptible.

88

Figure 6. Volcano plots depicting differential gene expression between treatments. A) Treated susceptible (S) relative to non-treated S

plants (SWT vs SWO), B) Treated tolerant (T) relative to treated S plants (TWT vs SWT), C) treated T relative to non-treated T plants

(TWT vs TWO), and D) treated T plants relative to non-treated S plants (TWO vs SWO). The x-axis shows the log fold change or

relative abundance. The P value (-log base 10) for differential gene expression is plotted on the y axis. Black dots represent genes that

did not change in expression; colored dots on the left indicate genes with significantly downregulated expression; colored dots on the

right indicates genes with significantly upregulated expression.

89

Figure 7. The number of differentially expressed genes common or specific to treated and non-

treated T and S plants.A 4-way Venn diagram depicting the distribution of differentially

expressed genes across all pairwise comparisons. The number within each shaded area is the

number of differentially expressed genes common between treatments.

90

Figure 8. Biological processes (A) and molecular functions (B) of upregulated genes in treated T

relative to treated S plants.

91

Figure 9. Biological processes (A) and molecular functions (B) of downregulated genes in

treated T relative to treated S plants.

92

Figure 10. Biological processes (A) and molecular functions (B) of differentially expressed

genes that are common in treated T relative to treated S and non-treated T plants.

93

Figure 11. Heat map analysis of genes that are putatively related to abiotic stress response in A.

palmeri.TWO (non-treated tolerant), SWO (non-treated susceptible), TWT (glufosinate-treated

tolerant), SWT (glufosinate-treated susceptible).

94

Figure 12. Gene expression fold-change of seven candidate NTS genes in glufosinate-tolerant

Amaranthus palmeri relative to –susceptible ones. Tolerant and susceptible plants from two

populations were identified based on the level of injury incurred after glufosinate treatment. GST

and CYP72A219 were highly expressed in the tolerant biotypes in at least one population. Non-

treated = before glufosinate treatment, Treated = 24 h after glufosinate treatment. Error bars

represent standard error from six biological replicates. HSP = heat shock protein, GST =

glutathione S-transferase, NMO = nitronate monooxygenase, ETF = ethylene-responsive

transcription factor, ABC = ABC transporter, NAC = NAC transcription factor, CYP72A219 =

cytochrome P450 CYP72A219.

95

CHAPTER IV

MULTIPLE RESISTANCE TO ALS-, PPO, AND EPSPS INHIBITORS IN

AMARANTHUS PALMERI FROM ARKANSAS, USA

96

ABSTRACT

Palmer amaranth (Amaranthus palmeri) is a major weed problem in agronomic crops in the

United States. The majority of the Palmer amaranth populations from Arkansas are resistant to

ALS inhibitors. Resistance to ALS inhibitors in Palmer amaranth populations is frequently

attributed to target-site mutation, however non-target-site based mechanisms can also be

involved. This study aimed to characterize the herbicide resistance profile and investigate target-

site and non-target-site resistance mechanisms to ALS inhibitors in a Palmer amaranth

population (15Cri-B) from Arkansas. Resistance level (R/S) for flumetsulam, imazethapyr,

trifloxysulfuron, fomesafen, and glyphosate were 4.8, 60.9, 4.4, 8.3, and 32 times, respectively,

for the 15Cri-B relative to the susceptible population. The addition of cytochrome P450 inhibitor

increased trifloxysulfuron phytotoxicity from 25% to 49% with malathion and to 66% with

piperonyl butoxide, suggesting the involvement of P450-mediated herbicide metabolism. A

Ser653Asn substitution in the ALS gene was detected in two out of seven resistant plants tested.

Resistant plants without any known resistance-conferring mutations had increased expression of

CYP81B and GSTF10 genes after trifloxysulfuron treatment, indicating their involvement in

metabolic resistance to ALS inhibitors. This Palmer amaranth population from Arkansas exhibit

both target-site and non-target-site based resistance to ALS inhibitors.

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INTRODUCTION

Palmer amaranth (Amaranthus palmeri S Watson) is a dioecious, aggressive weedy

species native to the area of the southwest United States and northern Mexico. It has become one

the most damaging and difficult-to-control weeds in major agronomic crops in the United States.

1Palmer amaranth is considered as an opportunistic, invasive and competitive weed due to its

rapid growth rate, continuous emergence period, high fecundity, genetic diversity, and its

capacity to evolve traits that increase its potential to grow and reproduce in various cropping

systems and environmental conditions.2,3 With its invasiveness and competitiveness, Palmer

amaranth can cause significant yield reduction in cotton (Gossypium hirsutum) (77%),4 soybean

(Glycine max) (79%)5, corn (Zea mays) (91%),6 grain sorghum (Sorghum bicolor) (63%),7 and

sweet potato (Ipomoea batatas) (94%).8 In addition to reducing yields, its large size (up to 3 m)

also decreases harvest efficiency as it hampers the operation of combines harvesters. Palmer

amaranth is an obligate outcrossing species which enables it to adapt and spread herbicide

resistance genes quickly. To date, various populations had evolved resistance to six different

mechanisms of action (MOA), namely: acetolactate synthase (ALS), 5-enol-pyruvylshikimate-3-

phosphate synthase (EPSPS), photosystem II, 4-hydroxyphenylpyruvate dioxygenase (HPPD),

microtubule, and protoporphyrinogen oxidase (PPO) inhibitors.9 Palmer amaranth populations in

Arkansas had evolved resistance to ALS inhibitors and glyphosate, and recently to PPO-

inhibiting herbicides which limits herbicide options for growers.10

Herbicides are a major tool in weed management; however, its usefulness is threatened

by the evolution of herbicide-resistant weeds. Herbicide resistance is an evolutionary adaptation

in weed species as a consequence of intensive herbicide selection pressure. Herbicide resistance

mechanism can be classified as either target-site (TSR) or non-target-site (NTSR). Target-site

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mechanisms include structural changes in the herbicide binding-site due to amino acid

substitution or increased expression of the target protein.11 Compared to TSR, NTSR is less often

reported and its molecular mechanisms are not well understood. With NTSR mechanism, the

number of herbicide molecules reaching the target site is minimized either by reduced herbicide

absorption or herbicide translocation or metabolism of the herbicide into non-toxic metabolites.

Reduction in herbicide absorption is due difference in cuticle properties or other structural

barriers that prevents that affects herbicide retention on the leaves and/or herbicide absorption

through the cuticle.11 Reduced herbicide translocation is characterized by restricted movement of

the herbicide within the plant, often due to sequestration of the herbicide into the vacuole or

inhibition of cellular transport.11-14

Herbicide detoxification usually involves oxidation of the herbicide molecules, usually

carried out by cytochrome P450 monooxygenases (P450s), conjugation of the activated

xenobiotic using thiols or sugars, transport of the conjugated molecules into the vacuole or

extracellular space by active transport, and degradation of the conjugated molecule into the

vacuole or extracellular space.15 NTSR mechanisms involve multiple genes and usually are part

of plant stress response.11 NTSR mechanisms pose a serious concern in weed management

because it can confer unpredictable resistance to a broad range of herbicides, including

herbicides that are yet to be commercialized.

Acetolactate synthase (ALS) inhibitors have been widely used for Palmer amaranth

control since their introduction in 1982.16 Inhibition of ALS enzyme affects the synthesis of

branched-chain amino acids (valine, leucine, and isoleucine), ultimately leading to plant death.

There are five chemical families of herbicide that inhibit ALS: sulfonylureas (SUs),

imidazolinones (IMIs), triazolopyrimidines (TPs), sulfonylaminocarbonyltriazolinones (SACTs),

99

pyrimidinylthiobenzoates (PTBs). The ALS herbicides are widely used because they provide

broad-spectrum weed control at low doses, soil residual activity, wide application windows,

excellent crop safety and low mammalian toxicity.17 However, these herbicides have high

propensity to select resistant weed populations due to strong selection pressure and high levels of

natural variability in the ALS gene.18 The widespread occurrence of Palmer amaranth populations

that had evolved resistance to ALS inhibitors has threatened the continued use of ALS-inhibiting

herbicides.

Most cases of ALS resistance are due to mutation in the ALS gene resulting in an enzyme

that is less sensitive to herbicide binding. To date, 29 amino acid substitutions in ALS that confer

herbicide resistance were identified at Ala122 (5), Ala205 (2), Arg377 (1), Asp376 (1), Gly654 (2),

Pro197 (11), Ser653 (3), and Trp574(4) in weed species.9 Substitution at Trp574 confers resistance to

IMIs, PTBs, SUs, and TPs,18 whereas Asp376Glu and Ala205Phe substitutions confer resistance to

all five chemical families of ALS inhibitors.19, 20 Amino acid substitution at Pro197 conferred

resistance to SUs with low or no cross-resistance to IMIs,18 whereas substitutions at Ala122 or

Ser653 conferred resistance to IMI herbicides with low-level resistance to SUs.21, 22 Recent

investigation on 20 Palmer amaranth populations from Arkansas revealed TSR mechanism

involving Trp574Ser mutation with a few cases of double mutations involving Ala122Thr,

Pro197Ala or Ser653Asn.23

Another way whereby plants can become resistant to ALS inhibitors is reducing the

amount of herbicide reaching ALS to below what is lethal (NTSR). Resistance to ALS inhibitors

is rarely attributed to reduced absorption and translocation,24-26 and in only few cases have they

been reported as partial-resistance mechanism.7, 28 Many studies have attributed enhanced

herbicide metabolism to the cytochrome P450 family of proteins by demonstrating that co-

100

application of the pesticide malathion, a P450 inhibitor, results in loss of resistance or reduced

level of resistance.29-32 An enhanced herbicide metabolism has been reported in ALS-resistant

rigid ryegrass (Lolium rigidum),33 wild mustard (Sinapis arvensis),34 and late watergrass

(Echinochloa phyllopogon).35, 36 Cytochrome P450 genes, CYP81A6 and CYP76B1, confer

resistance to sulfonylurea and phenylurea herbicides, respectively.37, 38 Multiple resistance

mechanisms may also exist in a weed population. Resistance to ALS inhibitor in Spanish corn

poppy (Papaver rhoeas) and flixweed (Descurainia Sophia L.) populations was controlled by

both target-site mutation (Pro197 substitution) and enhanced herbicide metabolism.39, 40

Several studies have identified the involvement of several NTSR genes in conferring

herbicide resistance by whole-transcriptome sequencing.39-42 With the availability of the Palmer

amaranth transcriptome,43 candidate NTSR genes conferring resistance to ALS inhibitors can be

validated. The objectives of this study were to determine the resistance profile of a Palmer

amaranth population from Arkansas to ALS herbicides, fomesafen, and glyphosate as well as to

unravel the resistance mechanism to ALS-inhibiting herbicides.

MATERIALS AND METHODS

Greenhouse experiments were conducted at Fayetteville, Arkansas. One known

susceptible (SS) and one ALS-resistant Palmer amaranth population (hereafter referred to as

15Cri-B) were used in the experiments. The SS seeds were collected from a vegetable field in

Crawford County, Arkansas and have been used routinely for resistance screening due to its

limited exposure to herbicides.44 The 15Cri-B population was collected from a soybean field in

the late summer of 2015. Little is known about the crop management history in this field, except

that the field has been planted with continuous soybean for several years. Inflorescence from at

101

least 10 female plants were collected, dried, threshed, and cleaned for bioassay in the

greenhouse.

Cross-resistance to multiple herbicides

Seeds were planted in 28- X 54- cm cellular trays filled with commercial potting soil

(Sunshine premix No. 1, Sun Gro Horticulture, Bellevue, WA). The experiment was set up in a

randomized complete block design with four replications. Each replication consisted of a tray

with 50 seedlings. Treatments included glufosinate at 549 g ha-1 (Liberty®, Bayer CropScience

LP, Research Triangle Park, NC), fomesafen at 263 g ha-1 (Flexstar®, Syngenta Crop Protection

LLC, Greensboro, NC) and glyphosate at 840 g ae ha-1 (Roundup POWERMAX®, Monsanto

Co., St. Louis, MO) and five ALS inhibitors namely; trifloxysulfuron at 8 g ha-1 (Envoke®,

Syngenta Crop Protection LLC, Greensboro, NC), pyrithiobac at 73 g ha-1 (Staple LX®, DuPont

Crop Protection, Wilington, DE), flumetsulam at 7 g ha-1 (Python®, Dow AgroSciences LLC,

Indianapolis, IN), imazethapyr at 70 g ae ha-1 (Pursuit®, BASF Corp., Research Triangle Park,

NC), and primisulfuron at 40 g ha-1 (Beacon®, Syngenta Crop Protection LLC, Greensboro, NC).

Pyrithiobac and trifloxysulfuron treatments included 0.25% non-ionic surfactant (NIS) (Induce®

non-ionic surfactant, Helena Chemical Co. Collierville, TN) by volume. Imazethapyr was

applied with 0.25% (v/v) NIS and 1.4% (w/v) ammonium sulfate (AMS), whereas primisulfuron

was applied with 0.25% (v/v) NIS and 2244 g AMS ha-1. Flumetsulam, fomesafen, and

glufosinate treatments included 0.5% (v/v) crop oil concentrate (COC) (Agri-Dex® crop oil

concentrate, Helena Chemical Co. Collierville, TN), 0.5% NIS and 3366 g AMS ha-1,

respectively. Herbicides were applied to 7.5-cm tall seedlings using a compressed air, motorized

boom, cabinet sprayer equipped with Teejet XR800067 flat fan nozzle (Teejet spray nozzles;

102

Spraying Systems Co., Wheaton, IL) calibrated to deliver 187 L ha-1 at 269 kPa. Mortality was

assessed 21 d after treatment (DAT) and each surviving plant was visually evaluated for injury

relative to the non-treated control. Injury was recorded on a scale of 0-100% where 0 had no

injury and 100% was dead. Data were analyzed using ANOVA in JMP Pro v.13.

Herbicide dose-response assay

Dose-response experiments were conducted as described by Salas et. al (2016).44 Seeds

were planted in 15-cm-diameter pots filled with commercial potting soil and thinned to 5 plants

per pot. The experiment was conducted in a completely randomized design with four

replications. Seedlings (7.5-cm tall) were treated with flumetsulam (1X = 7 g ha-1), imazethapyr

(1X = 70 g ae ha-1), trifloxysulfuron (1X = 8 g ha-1), glyphosate (1X = 840 g ae ha-1) and

fomesafen (1X = 263 g ha-1). The resistant plants were sprayed with eight herbicide doses

ranging from a 0X to a 8X dose; susceptible plants were ware sprayed with eight doses ranging

from 0X to 2X of the recommended herbicide dose. Visible injury and mortality were evaluated

21 DAT. Plants were cut at the soil surface, dried for 48 h, and weighed. Data were analyzed

using JMP Pro v. 13 in conjunction with SigmaPlot v. 13. Nonlinear regression analysis was

conducted and the data were fitted with a three-parameter log-logistic model (equation 1) to

determine the herbicide dose that would cause 50% control

𝑦 = 𝑐/[1 + 𝑒)−𝑎 (𝑥−𝑏) [1]

where y is the percent biomass reduction, a is the asymptote, b is the slope, c is the inflection

point, and x is the herbicide dose.

103

Whole-plant response to trifloxysulfuron with or without P450 inhibitors

Palmer amaranth plants (15Cri-B and SS) were grown in 15-cm diameter pots filled with

commercial soil mixture potting medium. Seedlings were thinned to 5 plants per pot after

emergence. Uniform sized-plants (7.5-cm tall) were treated with cytochrome P450 inhibitors

malathion (1000 g ha-1) (Hi-Yield Chemical Co., Bonham, TX), carbaryl (1100 g ha-1)

(Tessenderlo Kerley Inc., Phoenix, AZ) and piperonyl butoxide (1200 g ai ha-1) (MGK Co.,

Minneapolis, MN) 45 min before applying trifloxysulfuron at 8 g ha-1. Herbicide and P450

inhibitor applications were made using a laboratory sprayer equipped with a flat fan nozzle

(TeeJet spray nozzles, Spraying Systems Co., Wheaton, IL 60189) delivering 187 L ha-1. A

non-treated control was also provided for each population. At 21 DAT, plant injury was

evaluated visually. Aboveground biomass was harvested and dried at 60oC for 3 d. Results were

expressed as the percent biomass reduction relative to the non-treated control. The experiment

was conducted in a completely randomized design in a factorial treatment arrangement with

P450 inhibitor and herbicide as the main factors. Each treatment had four replications and the

experiment was conducted twice. Data were subjected to analysis of variance in JMP Pro v.13

software. Significant means were separated using Fisher’s protected LSD0.05.

ALS gene sequencing

Leaf tissues from four sensitive (SS) and seven trifloxysulfuron-resistant (15Cri-B) plants

were collected and stored immediately at -80oC freezer. Genomic DNA was extracted using the

modified hexadecytrimethylammonium chloride, previously described by Salas et al.44

Polymerase chain reaction (PCR) was conducted in a 40 µL mixture of 20 µL 2X PCR master

104

mix (Takara Bio USA Inc., San Francisco, CA), 2 µL of both forward and reverse primers (10

µM), 2 µL of genomic DNA (50 ng µL-1), and 16 µL water using MJ Research thermal cycler

(PTC-200, MJ Research, Inc., Waltham, MA). The following primers were used to sequence the

full ALS gene (2 kb): forward primer PAALS_F (5’-ATGGCGTCCACTTCAACAAAC-3’),

reverse primer PAALS_R (5’-GGTGATGGAAGAAGGGCTTATTAG-3); and internal primers

PAALS_F2 (5’-AGGATATTCCTAGAATTGTTAAGG-3’), PAALS_F3 (5’-

ATGCGGTTGTAAGTACCGGTGT-3’), PAALS_R1 (5’-CCTGGACCTGTTTTGATTGATA-

3’). PCR was performed under the following conditions; initial denaturation at 94°C for 2 min,

35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, extension at 72°C for 2

min, then final extension at 72°C for 10 min. The PCR amplification products were resolved on a

0.8% agarose gel to confirm the expected fragment size (2 kb). The PCR product was purified

using EZNA gel extraction kit (Omega Bio-Tek, Inc., Norcross, GA) following the

manufacturer’s instructions. The purified PCR product was sequenced at Eurofins Genomics,

Louisville, KY. The resulting DNA sequences were cleaned, aligned and analyzed using

Sequencher, BioEdit and ClustalW softwares.

Gene expression using qRT-PCR

The Palmer amaranth populations (15Cri-B and SS) were treated with trifloxysulfuron at

8 g ha-1 using the previously described procedure. Leaf tissues were collected 3 days before and

24 h after herbicide treatment. Genomic DNA from leaf tissues were subjected to PCR to amplify

the ALS gene following the previously described procedure. The purified PCR products were sent

for ALS gene sequencing. Plant samples which lacked any previously reported ALS point mutation

were used for the gene expression. The experiment was conducted in a completely randomized

105

design with herbicide treatment, plant biotype, and gene expression as the variables. Three

biological replicates from non-treated and treated samples of 15Cri-B and SS populations were

used. Total RNA was extracted from leaf samples using PureLink RNA Mini kit (Life

Technologies, Carlsbad, CA). cDNA synthesis was conducted using 5 µg total DNAse-treated

RNA using Reverse Transcription System first-strand cDNA synthesis kit (Promega). A 4-fold

serial dilution of cDNA samples (1:1, 1:5, 1:25, 1:125) was used to construct a standard curve.

Primers were designed to amplify ALS and six NTSR genes that were reported to be associated

with resistance to ALS-inhibiting herbicides (Table 1). DEAD-box RNA helicase and β-tubulin

were used as internal control for normalization of gene expression 45, 46

Quantitative real-time PCR (qRT-PCR) was conducted to determine changes in expression of

ALS and NTSR candidate genes relative to the reference genes. The qRT-PCR experiments were

conducted in a 12-µL volume containing 6.25 µL of SyberGreen Master Mix, 1 µL of 1:10 diluted

cDNA, and 0.5 µL of 10 µM primers (1:1 mix of forward and reverse primers). Amplification was

performed with three technical replicates using a Mastercycler EP Realplex machine (Eppendorf)

under the following conditions: 10 min at 94 oC, 40 cycles of 94 oC for 15 s and 60 oC for 1 min,

followed by a melt-curve analysis. Data were analyzed using Realplex 2.2 software. Fold-change

in gene expression as (2-ΔCt) was calculated by the comparative method,47 relative to the susceptible

samples, where ΔCT = [CT target gene –Ct mean of the two internal control genes]. Wilcoxon non-

parametric test (α=0.05) was used to determine statistical difference in gene expression between

resistant and susceptible biotypes.

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RESULTS AND DISCUSSION

Cross-resistance to ALS-, EPSPS- and PPO-inhibitors

The 15Cri-B population was resistant to ALS inhibitors flumetsulam, imazethapyr,

primisulfuron, pyrithiobac, trifloxysulfuron with mortality ranging from 0 to 35% (Table 2).

Acetolactate synthase inhibitors such as pyrithiobac and trifloxysulfuron were widely used in

cotton fields in the past to control broadleaf weed species including Palmer amaranth species.48,49

Similarly, imazethapyr used to be highly effective in managing Amaranthus species in soybean

fields.50,51 These once-effective herbicides now fail to provide the desired Palmer amaranth

control as observed in numerous fields. Cross-resistance to multiple ALS-inhibiting herbicides is

common in Palmer amaranth.2 Previous studies indicated imidazolinone-resistant populations

from Arkansas and Georgia that were also resistant to chlorimuron, diclosulam, and

pyrithiobac.52,53 Herbicide dose-response experiments conducted on whole plants confirmed

resistance to ALS inhibitors (flumetsulam, imazethapyr, trifloxysulfuron), fomesafen, and

glyphosate. Based on GR50, its resistance level was 4.8-, 60.9-, and 4.4-fold with flumetsulam

(triazolopyrimidine), imazethapyr (imidazolinone), and trifloxysulfuron (sulfonylurea),

respectively, relative to the SS population. The level of resistance in 15Cri-B is lower than what

has been previously reported for other populations of ALS-resistant Palmer amaranth

populations.54,55 A population of Palmer amaranth from Kansas and Argentina had >2,800- and

288-fold resistance to imazethapyr, respectively.54, 57 Resistant population of Palmer amaranth

from Mississippi and Georgia had 8- and 303-fold resistance to pyrithiobac, respectively.55, 56

The difference in resistance levels is partly due to the sensitivity of the standard susceptible

population used in greenhouse bioassay or due to differences in the mechanism of resistance or

107

both.58 Even though the15Cri-B population had a lower resistance ratio than other populations,

some plants survived at 8X the normal dose of flumetsulam, imazethapyr, and trifloxysulfuron.

The 15Cri-B population exhibited resistance not only to ALS inhibitors but also to

fomesafen (PPO inhibitor) and glyphosate (EPSPS inhibitor) herbicides. Fomesafen and

glyphosate had 77 and 61% control, respectively, of this population whereas glufosinate killed

91% of the population (Table 2). The resistance level was 8.3-fold with fomesafen and 32-fold

with glyphosate, relative to the SS population (Table 3). The mechanism of resistance to

fomesafen and glyphosate in 15Cri-B population is target-site based, via amino acid substitution

(Arg128Gly mutation in the PPO gene) and amplified EPSPS copy number, respectively.10, 59

Populations of Palmer amaranth resistant to both glyphosate and ALS inhibitors are prevalent

and have been identified in 10 states, including Arkansas, Arizona, Delaware, Florida, Georgia,

Illinois, Maryland, Michigan, Mississippi, Tennessee, and South Carolina.9,10,55,56 Other case of

multiple resistance was also documented such as the Palmer amaranth population from Michigan

that was resistant to ALS, EPSPS, and PSII inhibitors.58 Several Palmer amaranth populations in

Arkansas are resistant to glyphosate, ALS-, and PPO-inhibiting herbicides posing serious

challenge to growers.10, 60 With Palmer amaranth resistant to multiple modes of action, limited

herbicide options remain for its control. The use of photosystem II and long chain fatty acid

inhibitors for pre-emergence and foliar application of glufosinate in glufosinate-resistant crops

can be adopted. Herbicide-resistant trait technologies (LibertyLink®, Xtend®, or Enlist®) are

available and can be used along with integrated weed management practices such as applying

herbicides with different modes of action, crop rotation and other cultural and mechanical

practices (e.g. cover crops, tillage, hand weeding).61

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P450 inhibitor interaction with trifloxysulfuron

The involvement of P450 monooxygenase in metabolic resistance has often been detected

when specific P450 inhibitors enhance injury to resistant biotypes when applied in conjunction

with the herbicide.36, 62 The P450 inhibitors carbaryl, malathion, and piperonyl butoxide applied

alone caused little to no effect on the growth of Palmer amaranth. Therefore, these compounds,

by themselves, are not harmful to this weed. However, plants sprayed with malathion and

piperonyl butoxide prior to trifloxysulfuron treatment incurred higher phytoxicity compared to

plants treated with trifloxysulfuron alone (Table 4). Biomass reduction was 25% with

trifloxysulfuron alone but pretreatment with malathion or piperonyl butoxide increased Palmer

amaranth control to 49% and 66%, respectively. On the other hand, pretreatment with carbaryl

did not change the plant response compared to trifloxysulfuron alone. The increased efficacy of

trifloxysulfuron applied 45 min after malathion or piperonyl butoxide indicates P450-mediated

metabolism of the herbicide in resistant plants. Malathion is an excellent synergist for ALS-

inhibiting herbicides in ALS-resistant late watergrass, rigid ryegrass, and tall waterhemp

(Amaranthus tuberculatus).36, 62, 63 The synergistic interaction of malathion with sulfonylurea

herbicide is likely caused by competitive inhibition of cytochrome P450 degradation enzymes.64

The inhibition of herbicide activity by malathion occurs when atomic sulfur released from the

oxygenated organophosphate inhibits the P450 apoprotein.65 On the other hand, piperonyl

butoxide inhibits certain cytochrome P450 enzymes by causing steric hindrance created by its

long side-chain in the substrate access channel of the enzyme.38 Cytochrome P450 enzymes have

long been implicated in herbicide metabolism. These enzymes catalyze nonsynthetic

modifications, such as oxidation and hydroxylation.66 In fact, herbicide selectivity in weeds and

crops is attributed to the plant’s ability to metabolize the herbicide. Herbicide safeners protect

109

monocot crops from herbicide damage by inducing the production of P450 enzymes, glutathione

S-transferase, and glutathione peroxidase which can enhance the herbicide detoxification

processes.65 Hundreds of P450 genes exist in plants genomes, and each P450 participates in

various biochemical pathways to produce primary and secondary metabolites.67 The complexity

of cytochrome P450s increases the difficulty of elucidating the genetics of resistance, however,

the use of advanced molecular biology and genomics approaches are beginning to provide new

information on the identity and role of P450 genes in endowing metabolic resistance in plants.

Sequence analysis of acetolactate synthase gene

Previous researches have shown that amino acid substitution in the ALS gene is the most

common resistance mechanism in ALS-resistant Amaranthus species18, 23, 68 The ALS gene of

seven resistant plants from 15Cri-B contained nucleotide changes at multiple loci; however most

of the polymorphisms were silent mutations. A guanine to adenine change at position 653 was

detected in two resistant plants which resulted in Ser653Asn amino acid mutation (number

relative to Arabidopsis) (Fig. 1). The other five resistant plants did not have any of the known

resistance-conferring mutations to ALS herbicides. Variability in the ALS gene within a

population was also reported in ALS-resistant Palmer amaranth from Argentina in which five

allelic versions of the ALS gene were detected.57 Given the high genetic diversity of Palmer

amaranth, it is not surprising to have several genotypes of Palmer amaranth in a population. Our

results agree with the findings of other researchers, who also reported that Ser653Asn mutation

conferred high-level resistance to imidazolinone herbicides in tall waterhemp and downy brome

(Bromus tectorum).69,70 In greenhouse bioassays 15Cri-B had high level of resistance to

imazethapyr (60.9-fold), and low level of resistance to trifloxysulfuron (4.4-fold) and

110

flumetsulam (4.8-fold). The Ser653Asn mutation has been almost exclusively linked to

imidazolinone resistance, conferring low resistance to sulfonylurea in some cases,18 however,

15Cri-B was also cross-resistant to TPs (flumetsulam) and PTBs (pyrithiobac). NTSR

mechanisms possibly exist in these plants that would enable broad resistance to ALS herbicides

belonging to different chemical families. Furthermore, the presence of plants that carry the

Ser653Asn mutation and plants that do not harbor ALS mutation suggest the existence of both TS

and NTSR mechanisms in 15Cri-B population.

ALS and NTSR gene expression analysis

Target gene overexpression has been reported to confer resistance to herbicides.

Glyphosate-resistant Palmer amaranth populations contain multiple copies of the EPSPS gene

resulting in overproduction of the target enzyme EPSPS.71-73 In our experiment, ALS-resistant

and –susceptible Palmer amaranth biotypes had similar ALS transcript abundance (1-fold)

suggesting that the ALS gene is not overexpressed in resistant plants. The ALS gene expression

did not differ before and after trifloxysulfuron treatment in both biotypes. Stable expression of

the ALS gene has been documented; in fact, it has been used as reference in several gene

expression.72, 74 However, in shortawn foxtail (Alopecuros aequalis), the ALS copy number is

variable.75 The ALS copy number variation in shortawn foxtail is not likely an adaptation for

herbicide resistance since the additional ALS copies were deemed to be non-functional

pseudogenes. In the case of Palmer amaranth, the ALS gene was not differentially expressed

between ALS-resistant and –susceptible plants, which indicates that other mechanism(s), apart

from target gene overexpression, endow resistance to other ALS inhibitors.

111

The expression patterns of six genes [four cytochrome P450s (CYP72A, CYPA219A,

CYP81D, CYP81B), one glycosyltransferase (GT), and one glutathione-S-transferase (GSTF10)]

were examined before and after trifloxysulfuron treatment in resistant and susceptible plants.

CYP81B and GSTF10 were induced upon trifloxysulfuron application and were not

constitutively expressed in non-treated plants (Fig. 2). The expression of GSTF10 and CYP81B

in resistant plants ranged from 3- to 5-fold relative to the susceptible plant suggesting their

involvement in NTSR to trifloxysulfuron. Cytochrome P450 and glutathione-S-transferase have

been implicated in herbicide degradation; the former catalyzes herbicide oxidation and the latter

facilitates conjugation with the herbicide metabolite.11

The biochemical role of cytochrome P450-mediated herbicide metabolism have been

reported in herbicide-resistant rigid ryegrass and late watergrass populations.42, 62,76,77,79 For

example, high expression of CYP81B1, CYP81A21, and CYPA12 were associated with

resistance to SUs and TPs in Lolium and late watergrass populations.77,79,80 Several other

cytochrome P450 genes have been linked to ALS resistance in various weed populations such as

CYP72A254 (also known as CYPA72A219) in late watergrass (bispyribac),78 CYP81D in black-

grass (Alopecurus myosuroides) (mesosulfuron and iodosulfuron),81 CYP94A1, CYP94A2,

CYP71A4 and CYP734A6 in shortawn foxtail (mesosulfuron) (Table 5).41 In addition, many

CytP450s conferring resistance to ALS herbicides have been documented in several major crop

species, such as CYP81A6 and CYP72A31 in rice (Oryza sativa) and CYP81A9 in maize (Zea

mays) (Table 5).37,82,83 It was demonstrated that the wheat (Triticum aestivum) CYP71C6v1

expressed in yeast (Saccharomyces cerevisiae) is able to metabolize several sulfonylurea

herbicides such chlorsulfuron and triasulfuron through phenyl ring hydroxylation.84 The

112

CYP81B gene identified in Palmer amaranth (15Cri-B) may also confer resistance to multiple

herbicides, but this will require further investigation.

Glutathione S-transferases (GSTs) are ubiquitous in plants and have defined roles in

herbicide detoxification.85 Differences in glutathione availability and in the portfolio of GST

isoenzymes are associated with herbicide selectivity and herbicide resistance.86 For example,

tolerance of maize and giant foxtail (Setaria faberi) to atrazine is due to the high levels of GST

that facilitate the detoxification of the herbicide via glutathione conjugation.86, 87 Furthermore,

induction of glutathione and GSTs by herbicide 'safeners' is employed commercially to protect

crop plants from injury by certain herbicides.88 GSTs (phi and tau class) were involved Lolium

resistance to diclofop.89 Furthermore, GSTA and GST1 were also recently reported to participate

in non-target resistance or tolerance to ALS inhibitors in Lolium and maize, respectively.82,90 It

was once demonstrated that transgenic tobacco plants (Nicotiana tabacum) expressing maize

GST1 had enhanced detoxification of the herbicide alachlor.91 In ACCase-resistant black-grass,

GSTF1 catalyzed the conjugation of herbicide to glutathione as well as acted as peroxidase

protecting the cell from oxidative damage.92-94 Transgenic Arabidopsis (Arabidopsis thaliana)

expressing the black-grass GSTF1 gene (AmGSTF1) has improved tolerance to alachlor and

atrazine due to increased accumulation of protective flavonoids.94 Glutathione S-transferases

contribute herbicide resistance by detoxifying and transporting herbicide metabolites and/or

counteracting oxidative stress. It remains to be investigated whether the GSTF10 of Palmer

amaranth is involved in herbicide conjugation or protection from oxidative damage. An ortholog

of GSTF10 in Arabidopsis (AtFSTF10) possesses only transferase activity and not peroxidase

activity.95 Thus, Palmer amaranth GST10 is likely to be involved in detoxification of ALS-

inhibiting herbicides by herbicide conjugation with glutathione.

113

Our study showed ALS target-site mutation (Ser653Asn) conferring resistance to

trifloxysulfuron in ~29% (2/7) of 15Cri-B Palmer amaranth population. Previous reports

associated Ser653Asn mutation with high-level resistance to IMIs and low-level resistance to

SUs,18 however, the 15Cri-B population exhibited resistance not only to IMIs and SUs but to

PTBs and TPs. It is likely that resistant plants that carried the ALS Ser653Asn mutation may

harbor NTSR mechanisms, but this needs to be verified. The majority of the resistant plants did

not carry the Ser653Asn mutation but showed induction of NTSR (GSTF10 and CYP81B) genes,

which would endow resistance to multiple ALS herbicides. The occurrence of both TSR and

NTSR to ALS inhibitors in the same population was also reported in a Palmer amaranth from

Kansas.96 In that study, 30% of the chlorsulfuron-resistant plants harbored ALS Pro197Ser

mutation and the remaining 70% was deduced to have cytochrome P450-mediated metabolism

based on a malathion pretreatment assay.96 Other weed species displaying both TSR and NTSR

to ALS inhibitors include corn poppy,40 flixweed,39 and rigid ryegrass populations.39,40,97 The

evolution of TSR and NTSR in weed species predisposes resistance to multiple herbicide

families and modes of action. With Palmer amaranth being a prolific and obligate crossing

species, plants in a population can accumulate multiple resistance mechanisms which would

likely drive resistance evolution faster and is therefore detrimental to chemical weed

management.

114

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121

Table 1. Primers used in the gene expression analysis by qRT-PCR.a

Gene Primer Sequence Name Reference

Cytochrome P450

CYP72A219

GGACAAGAAACTACATCGACA Cyp72A219_F3 78

TGTTTGCAGGACTTCTTCC Cyp72A219_R3

Cytochrome P450

CYP81D

GTTTATACCATTCGGGTCAGG Cyp81D_F4 80

TTCTCCCATTCTTTCCCATTC Cyp81D_R4

Cytochrome P450

CYP81B

AACGACTTCTAGCACAATGG Cyp81B_F2 76

GTCCTACGTGCTCTAAGATTTC Cyp81B_R2

UDP-

glycosyltransferase

(GT)

GAATTGATGGAGGGAAGGAATG GT_F3 39,76

CCTTCAACTTTAGTAGCCTCTTG GT_R3

Glutathione-S-

transferase

(GSTF10)

GCTATTTAGCTGGAGACGAC GST_F1 76

CCCACCATCTACTTACATTCTC GST_R1

Cytochrome P450

CYP72A215

TGCACAATCTCTCGAATGG Cyp72_F2 76,83

GGATCGTGGTGGGTATAAAG Cyp72_R2

ALS GCTGCTGAAGGCTACGCT ALS_F2 72

GCGGGACTGAGTCAAGAAGTG ALS_R2

DEAD-box RNA

helicase

TTGGAACTGTCAGAGCAACC A36_F 45

GAACCCACTTCCACCAAAAC A36_R

β-tubulin ATTCCCTCGGCTTCATTTC BT_F1 46

TCCCACATTTGCTGAGTTAG BT_R2

a Herbicide-treated and non-treated Palmer amaranth plants (15Cri-B and SS populations) with

three replications per treatment were used for the qPCR assay. The herbicide-treated plants were

harvested 24 h after application of trifloxysulfuron at 8 g ha-1.

122

Table 2. Response of 15Cri-B Palmer amaranth population to the normal dose of ALS

(flumetsulam, imazethapyr, primisulfuron, pyrithiobac, trifloxysulfuron), fomesafen, glyphosate

and glufosinate herbicidesa.

Mode of Action Herbicide Mortality

(%)

Acetolactate synthase (ALS) inhibitor

Flumetsulam 35

Imazethapyr 21

Primisulfuron 4

Pyrithiobac 0

Trifloxysulfuron 3

Protoporphyrinogen oxidase (PPO) inhibitor Fomesafen 77

Enolpyruvyl shikimate-3-phosphate synthase

(EPSPS) inhibitor

Glyphosate 61

Glutamine synthetase (GS) inhibitor Glufosinate 91

a Flumetsulam (7 g ai ha-1) , imazethapyr (70 g ae ha-1), primisulfuron (40 g ai ha-1), pyrithiobac

(73 g ai ha-1), trifloxysulfuron (8 g ai ha-1), fomesafen (263 g ai ha-1), glyphosate (840 g ae ha-1),

and glufosinate (549 g ai ha-1) were applied to 7.5-cm tall seedlings. Each herbicide was applied

to 200 seedlings.

123

Table 3. Level of resistance to ALS, PPO, and EPSPS inhibitors in 15Cri-B Palmer amaranth

population.

Mode of Action Herbicide GR50a

(g ai ha-1)

R/Sb

SSc 15Cri-B

Acetolactate synthase

(ALS) inhibitor

Flumetsulam 1.68

(1.04-2.32)d

8.16

(5.24-11.08)

4.8

Imazethapyr 6.42

(4.45-8.39)

67.35

(48.27-86.43)

60.9

Trifloxysulfuron 0.85

(0.63-1.07)

3.8

(2.75-4.87)

4.4

Protoporphyrinogen

oxidase (PPO)

inhibitor

Fomesafen 15.10

(10-21)

125.26

(85-164)

8.3

Enolpyruvyl

shikimate-3-

phosphate synthase

(EPSPS) inhibitor

Glyphosate 11.9

(9.0-14.7)

385

(287-410)

32

a Herbicide dose required to cause 50% biomass reduction. b Resistance level (R/S) calculated using the GR50 of the resistant population relative to the

susceptible population. c SS = susceptible standard population d Values inside the parenthesis denote 95% confidence intervals.

124

Table 4. Effect of pretreatment with P450 inhibitorsa on the phytotoxicity of trifloxysulfuron (8 g

ha-1) on 15Cri-B Palmer amaranth population.

P450 inhibitor Biomass reduction relative to the non-

treated control (%)

No herbicide With

trifloxysulfuron

Carbaryl 0 22

Malathion 5 49

Piperonyl butoxide 0 66

No P450 inhibitor 0 25

LSD0.05

Main effect of P450 inhibitor 10

Main effect of herbicide 14

P450 inhibitor x herbicide interaction 16

aP450 inhibitors were applied 45 min before trifloxysulfuron treatment.

125

Table 5. Cytochrome P450 genes involved in NTSR to ALS inhibitors in various weed and crop

species.

Cytochrome P450 gene

Species Herbicide Reference

CYP71A Alopecurus myosuroides iodosulfuron-methyl,

mesosulfuron-methyl

81

CYP71A4 Alopecuros aequalis mesosulfuron-methyl 41


CYP72A254 Echinochloa phyllopogon bispyribac-sodium 78

CYP71B Alopecurus myosuroides iodosulfuron-methyl,

mesosulfuron-methyl

81

CYP71A31 Oryza sativa bispyribac-sodium 83

CYP71C6v1 Triticum aestivum chlorsulfuron, triasulfuron 84

CYP81A6 Oryza sativa bensulfuron-methyl 37

CYP81A9 Zea mays nicosulfuron 82

CYP81A12 Echinochloa phyllopogon bensulfuron-methyl, penoxsulam 80

CYP81A21 Echinochloa phyllopogon bensulfuron-methyl, penoxsulam 80

CYP81B1 Lolium pyroxsulam, iodosulfuron-methyl,

mesosulfuron-methyl

79

CYP81D Alopecurus myosuroides iodosulfuron-methyl,

mesosulfuron-methyl

81



126

Figure 1. Amino acid sequence alignment of acetolactate synthase (ALS) in sensitive (SS) and

resistant (15Cri-B) Palmer amaranth plant samples. Two resistant plants harbored Ser653Asn

(AGC to AAC) mutation.

127

Figure 2. Gene expression analysis of ALS and NTSR genes in trifloxysulfuron-resistant (15Cri-

B) and –susceptible (SS) Palmer amaranth biotypes. Fold-change (2-ΔCt) in gene expression was

calculated relative to the susceptible samples, where ΔCt = [Ct target-gene - Ct mean of internal

control genes]. Means and standard errors from three biological replicates are shown. Wilcoxon

non-parametric test was used to compare differential gene expression between ALS-resistant and

susceptible plants. An asterisk denotes fold change significance at P<0.05.

128

CHAPTER V

RESISTANCE TO PPO-INHIBITING HERBICIDE IN PALMER AMARANTH FROM

ARKANSAS

© 2016 Pest Management Science published by JohnWiley & Sons Ltd on behalf of Society of

Chemical Industry.

129

Abstract

BACKGROUND: The widespread occurrence of ALS inhibitor- and glyphosate-resistant

Amaranthus palmeri has led to increasing use of protoporphyrinogen oxidase (PPO)-inhibiting

herbicides in cotton and soybean. Studies were conducted to confirm resistance to fomesafen (a

PPO inhibitor), determine the resistance frequency, examine the resistance profile to other foliar-

applied herbicides, and investigate the resistance mechanism of resistant plants in a population

collected in 2011 (AR11-LAW B), and its progenies from two cycles of fomesafen selection (C1

and C2).

RESULTS: The frequency of fomesafen-resistant plants increased from 5% in the original

AR11-LAW-B to 17% in the C2 population. The amounts of fomesafen that caused 50% growth

reduction were 6-, 13-, and 21-fold greater in AR11-LAW-B, C1, and C2 populations,

respectively, than the sensitive ecotype. The AR11-LAW-B population was sensitive to atrazine,

dicamba, glufosinate, glyphosate, and mesotrione but resistant to ALS-inhibiting herbicides

pyrithiobac and trifloxysulfuron. Fomesafen survivors from C1 and C2 populations tested

positive for the PPO glycine 210 deletion previously reported in waterhemp (Amaranthus

tuberculatus).

CONCLUSION: These studies confirmed that Palmer amaranth in Arkansas has evolved

resistance to foliar-applied PPO-inhibiting herbicide.

130

INTRODUCTION

Palmer amaranth (Amaranthus palmeri S. Watson) is one of the most common,

troublesome, and economically damaging agronomic weeds throughout the southern United

States1. This weed continues to emerge throughout the summer making control critical from crop

emergence to harvest. The competitive ability of Palmer amaranth is attributed to its fast growth

rate2, high fecundity3, good light interception, and high water use efficiency4. With estimates of

over 600,000 seeds per plant, it can replenish the seedbank3 in one generation. Because it is

highly competitive with crops, it can reduce crop yield. Palmer amaranth densities of 8 and 9

plants m-2 can reduce soybean yield by 78%5 and corn grain yield by 91% 6, respectively. Fast et

al. (2009)7 reported that Palmer amaranth interference for 63 d after crop emergence caused 77%

cotton yield loss. Apart from reducing yield, more severely in stripper cotton, high infestation of

Palmer amaranth interferes with cotton harvest and can increase harvest time by two- to four-

fold8.

Palmer amaranth control has become a challenge because of its high propensity to evolve

herbicide resistance, resulting in reduced herbicide options in infested crops such as cotton and

soybean. To date, Palmer amaranth has been confirmed resistant to herbicides spanning five

modes of action: acetolactate synthase (ALS) inhibitors, carotenoid biosynthesis (4-

hydroxyphenylpyruvate dioxygenase) inhibitors, enolpyruvyl shikimate-3-phosphate synthase

inhibitor (glyphosate), mitosis inhibitors (dinitroanilines), and photosystem II inhibitors

(triazines)9. Being dioecious, Palmer amaranth is an obligate cross-pollinated species, allowing

herbicide resistance to spread rapidly10. Sosnoskie et al. (2012)11 reported that glyphosate

resistance trait was transferred across a distance of at least 300 m through pollen flow.

131

The widespread occurrence of resistance to ALS inhibitors and glyphosate in Palmer

amaranth has led to increasing use of protoporphyrinogen oxidase (PPO)-inhibitor herbicides

such as fomesafen, flumioxazin, saflufenacil, and sulfentrazone. Advantageous characteristics of

PPO inhibitors include broad herbicidal spectrum as these are active against many

monocotyledon and dicotyledon weeds, have low mammalian toxicity, low effective rates, rapid

onset of action, and long residual activity on some herbicides in this group12. The PPO enzyme

catalyzes the conversion of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto

IX) which is the last common step in the biosynthesis of heme and chlorophyll13. In plants, two

PPO isoforms are encoded by two different PPO nuclear genes, PPX1 and PPX2. These isoforms

share little sequence identity (25%) and differ in their subcellular targeting. PPO1 and PPO2 are

localized in plastids and mitochondria, respectively; however, at least some PPO2 isoforms are

dual-targeted to both organelles14-16. Inhibition of PPO by herbicides results in the generation of

singlet oxygen species that attack lipid and protein membranes leading to plant death17.

The PPO enzyme is inhibited by several herbicide chemical classes (e.g. diphenyl ethers,

heterocyclic phenyl ethers, oxadiazoles, phenyl imides, triazolinones and pyrazoles). Herbicides

that inhibit PPO have been in the market since the 1960s and were primarily used in soybean.

One of these is fomesafen, a diphenyl ether herbicide, which can be applied preplant,

preemergence or postemergence for control or suppression of broadleaf weeds, grasses and

sedges in soybean. Resistance to PPO herbicides has been slow to evolve (about four decades

from first commercialization), and to date, has been confirmed only in seven weed species9. The

first weed to evolve resistance to PPO herbicides was waterhemp (Amaranthus tuberculatus) in

200118. Resistance to PPO herbicides in weedy species has been attributed to target-site mutation

in the PPX2 gene19-20. A unique target-site amino acid deletion (Gly210) and Arg98Leu

132

substitution confers PPO resistance in waterhemp19 and common ragweed (Ambrosia

artemisiifolia)20, respectively.

PPO herbicides are widely utilized for controlling glyphosate-resistant Palmer amaranth

in conventional and RoundupReady® soybeans and in cotton. Intensive use of herbicides exerts

high selection pressure leading to the evolution of herbicide-resistant weed populations. This

paper describes fomesafen-resistance in Palmer amaranth populations from Arkansas.

2 MATERIALS AND METHODS

2.1 Plant materials

In the late summer of 2011, Palmer amaranth samples escaping from either glufosinate or

PPO-inhibiting herbicide applications were collected from several fields in Arkansas.

Inflorescences from 10 female Palmer amaranth plants per field were collected, dried, threshed,

and cleaned for bioassay in the greenhouse. One such field in Lawrence County has been planted

with LibertlyLink® soybean and RoundupReady® corn in alternate years since 2011 and treated

with either fomesafen and glufosinate or glyphosate. Fomesafen was applied during the soybean

production year. Five-hundred mg of seeds from each of the 10 plants per field were mixed to

make a composite, which was used for subsequent experiments. Susceptible Palmer amaranth

seeds (SS) were collected from a vegetable field in Crawford County, Arkansas. This vegetable

field was not exposed to glyphosate or PPO-inhibiting herbicides.

Plants were grown in a greenhouse maintained at 32/25 °C ± 3 °C day/night temperature

with a 16-h photoperiod. Plants were watered daily and fertilized with MiracleGro®, a water-

soluble all-purpose plant food containing 15-30-15% NPK, every two weeks.

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2.2 Fomesafen resistance bioassay in the greenhouse

Palmer amaranth seeds were planted in 28 x 54 cm cellular trays (Redwayfeed Garden

and Pet supply, 290 Briceland Rd, Reedway, CA 95560) using Sunshine® premix soil (Sunshine

premix #1®, Sun Gro Horticulture, 15831 NE 8th Street, Suite 100, Bellevue, WA 98008). The

experiment was conducted in a randomized complete block design with five replications. Each

replication consisted of one cellular tray with 50 seedlings, grown at one seedling per cell. The

test was repeated. Thus, from each composite seed sample, a total of 500 plants were sprayed

with the recommended dose of fomesafen at 264 g ha-1 (Flexstar 1.88 EC, Syngenta) when

seedlings were 7.5-9 cm tall. The herbicide was applied with 0.5% by volume nonionic

surfactant (NIS), using a laboratory sprayer equipped with a flat fan spray nozzle (TeeJet spray

nozzles, Spraying Systems Co., Wheaton, IL) delivering 187 L ha-1 at 269 kPa. After 21 d, the

overall effects of fomesafen (stunting, chlorosis, necrosis, and desiccation) were assessed

visually relative to the nontreated control using a scale of 0 to 100 where 0 = no visible injury

and 100 = complete death.

Survivors from fomesafen treatment were grown, and allowed to interbreed, to produce

C1 and C2 populations of progenies. A subset of nine survivors (which eventually consisted of 3

females and 6 males) from AR11-LAW-B produced the C1 population. C1 plants were sprayed

with fomesafen following the same procedure described previously. A subset of six survivors

(which eventually consisted of 1 female and 5 males) from C1 plants were grown for seed

production to produce C2 population.

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2.3 Progression of resistance from the original AR11-LAW-B to C2 Palmer amaranth

population

Seeds from the C1 and C2 Palmer amaranth populations were planted in cellular trays to

determine the frequency of fomesafen-resistant plants and the progression of resistance from the

original population to the C2 population. The experiment was conducted twice in a randomized

complete block design with 5 replications; the C1 population was bioassayed four times. In each

cellular tray or replication, 50 plants were grown separately and sprayed with 264 g ha-1 of

fomesafen using the herbicide-application method described previously. Mortality and plant

injury were recorded at 21 DAT.

2.3 Fomesafen dose-response bioassay

Palmer amaranth seeds of the susceptible, original AR15-LAW-B, C1, and C2

populations were planted in 11 x 11 cm square pots filled with Sunshine Mix LC1 potting soil

(Sun Gro Horticulture Canada Ltd., Vancouver, British Columbia, Canada). Seedlings were

thinned to five per pot. Seedlings at 7.5-9 cm tall were sprayed with eight doses of fomesafen

from 0 to 2109 g ha-1, which corresponds to 0 to 8 times the recommended field dose. The SS

population was sprayed with 7 doses from 4 to 264 g ha-1 corresponding to 1/64 to 1X

recommended dose, with a nontreated check. The herbicide was applied with a 0.5% NIS as

described in Section 2.2. The experiment was conducted twice in randomized complete block

design with five replications. At 21 DAT, visible injury and the number of survivors were

recorded. The above-ground plant tissue was harvested, placed in brown paper bag, dried at 60°C

for 48 h, and dry weights recorded. Data were expressed as percentage of biomass reduction

relative to the nontreated control. The biomass data generated from two runs were pooled as the

test for homogeneity of variance showed that the variance across runs was similar. Regression

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analysis was conducted using SigmaPlot v.13. The percent biomass reduction and mortality were

fitted to nonlinear, sigmoid, three-parameter Gompertz regression model defined by (Equation

1),

y = a*exp [-exp {-b*(x-c)}] [1]

where Y is the biomass reduction expressed as a percentage of the nontreated control or

mortality percentage; a is the asymptote; b is the growth rate; c is the inflection point; and x is

the fomesafen dose. The dose needed to kill 50% (LD50) of the population or cause 50% biomass

reduction (GR50) was calculated from the above equation.

2.4 Response of Palmer amaranth population to other foliar-applied herbicides

Palmer amaranth seeds from the susceptible and original AR11-LAW-B populations were

planted in cellular trays in the greenhouse. Uniform-sized plants (7.5-9 cm tall) were treated with

atrazine at 2244 g ha-1, dicamba at 280 g ha-1, glufosinate at 547 g ha-1, glyphosate at 870 g ha-1,

mesotrione at 105 g ha-1, and ALS inhibitors. Glufosinate and mesotrione treatments included

3366 g ammonium sulfate (AMS) ha-1 and 1% crop oil concentrate (COC), respectively. The

ALS inhibitors and their respective rates included pyrithiobac at 73 g ha-1 and trifloxysulfuron at

8 g ha-1, applied with 0.25% NIS by volume. Herbicide treatments were applied as described in

Section 2.2. Following application, plants were placed on greenhouse benches in a randomized

complete block design. Each treatment was replicated twice, with each replication consisting of

50 plants. Mortality was assessed at 21 DAT.

Because AR11-LAW-B was found resistant to pyrithiobac and trifloxysulfuron, dose-

response assays were conducted to determine the level of resistance to these herbicides. Seeds

were planted in 13-cm round pots filled with commercial potting soil and seedlings were thinned

136

to five plants per pot. The SS population was sprayed with 8 herbicide doses from 1/16X to 4X

the recommended dose of pyrithiobac (1X = 73 g ha-1) and trifloxysulfuron (1X = 8 g ha-1),

including a nontreated check. AR11-LAW-B population was treated with eight doses of

pyrithiobac (0 to 1166 g ha-1) and eight doses of trifloxysulfuron (0 to 31 g ha-1), which

corresponds to 0 to 16X the recommended herbicide dose. Herbicides were applied following the

procedure described previously. The experiment was conducted in a completely randomized

design with five replications. At 28 DAT, plants were cut at the soil surface, stored in the dryer

for 2 d, and the dry weights recorded. Percent biomass reduction relative to the nontreated

control were fitted to nonlinear, sigmoid, three-parameter Gompertz regression model. The dose

needed to reduce the aboveground biomass by 50% was obtained from the above equation using

SigmaPlot v. 13.

2.5 Mechanism of resistance in PPO-resistant Palmer amaranth

Fomesafen survivors from C1 and C2 populations were tested for the presence of the

PPO glycine 210 deletion (ΔG210). This deletion confers resistance to PPO herbicides in

waterhemp, a relative weedy species of Palmer amaranth19. Young leaf tissues from 81 C1 and

13 C2 plants that survived the application of 264 g fomesafen ha-1 were collected and stored at -

80°C. Tissues from three sensitive plants (SS) were also collected for DNA extraction. Genomic

DNA from 100 mg leaf tissue was extracted using hexadecytrimethylammonium bromide

(CTAB) method21 following the modification of Sales et al22. The extracted genomic DNA was

quantified using a NanoDrop spectrophotometer model ND-1000 (Thermo Scientific,

Wilmington DE).

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The ΔG210 codon deletion was detected using an allele-specific PCR assay as previously

described for waterhemp23. The same assay was predicted to work for Palmer amaranth based on

PPX2 sequence data previously generated from this species24.

3 RESULTS AND DISCUSSION

3.1 Progression of PPO-resistance in PA-AR11-LAW-B population

The frequency of fomesafen-resistant plants increased from 5% in the original AR11-

LAW-B to 17% in the C2 population, in response to the 264 g ha-1 dose of fomesafen. Of the 500

plants treated, 25 survived in the original population. The number of survivors increased in the

C1 (n=56) and C2 (n=86) population. By the practical description of a resistant population25, the

field population in 2011 (5% resistant individuals) would still be considered a susceptible

population. The few remnant plants in the field did not cause any economic loss nor were noticed

by the farmer. Until the number of resistant plants reach a level that presents a problem for

management and yield loss, the population would not be considered resistant25. This current

work showed that once a few individuals carrying the resistance trait is selected, sustained

herbicide selection pressure on a prolific species like Palmer amaranth could produce a resistant

population in two years. This would be when the farmer would call for assistance from crop

advisors or Extension Service (personal experience). By the time a population-level resistance is

noticed, it is already too late for that field; by then the resistant plants would have had already

deposited a large amount of seeds in the soil and the resistant allele(s) would remain in the

population.

Not all progenies of fomesafen-resistant survivors carried the PPO-mutation as shown by

the resistance frequencies. This suggests that the resistance trait is segregating and progenies are

heterogeneous. Palmer amaranth has wide genetic variability owing to its cross-pollinating

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behavior. However, for every round of selection, the population becomes more homogeneous.

The C2 population had more frequency of resistant plants than C1 or its parent population AR11-

LAW-B. After two cycles of selection, the frequency of resistant plants increased about three

times. There are examples of rapid evolution of herbicide resistance in response to intense and

sustained selection pressure26. The Weed Science Society of America (WSSA) defines herbicide

resistance as the inherited ability of a plant to survive and reproduce following exposure to a

dose of herbicide that is normally lethal to the wild type27. In herbicide resistance, there is a

change in the weed population response (i.e. reduced efficacy of the herbicide) with time as

observed with AR11-LAW-B. Thus, the field-collected sample in 2011 was in the early state of

resistance evolution to PPO herbicides. The large-scale testing of our Palmer amaranth collection

(2008 – 2011) for differential tolerance to fomesafen was conducted in 2013. The tests for

heritability and confirmation of G210 deletion among fomesafen survivors were completed in

2015. Thus, the detection and confirmation of resistance to PPO herbicides in Palmer amaranth

happened years later from collection of the resistance-bearing population.

The farmer of AR11-LAW-B field adopted a corn-soybean crop rotation system. In 2015,

the field was clean, except for a sparse remnant Palmer amaranth (about 1 ha-1). Three plants

were sampled and tested for resistance to fomesafen; one of these tested positive for the G210

deletion mutation. This demonstrates that once the resistant allele has been selected for, it could

remain in the population unless the seedbank is depleted.

3.2 Resistance level to fomesafen

Increasing the dose of fomesafen reduced the dry weight of SS, AR11-LAW-B, C1, and

C2 populations. The fomesafen dose that caused 50% growth reduction or GR50 (+ 1 standard

error) was 13 (+ 0.86), 82 (+ 6.1), 168 (+ 11.9), and 265 (+ 20.4) g fomesafen ha-1 for SS, AR11-

139

LAW-B, C1, and C2 population, respectively (Figure 1, Table 1). Based on these GR50 values,

the level of resistance to fomesafen in AR11-LAW-B, C1, and C2 population was 6-, 13-, and

21-fold relative to the sensitive population (SS). The GR50 increased from 82 g ha-1 in the

original AR11-LAW-B to 265 g fomesafen ha-1 in the C2 population. The commercial field dose

of fomesafen (264 g ha-1) is required to reduce aboveground biomass by 50% in the C2

population indicating that the normal field dose would no longer provide effective control. After

two more cycles of selection from the point where 5% resistant individuals were detected, the

GR50 increased three times, reflecting the observed increase in the frequency of resistant plants.

The herbicide dose that caused 50% mortality (LD50) was 16 (+ 1.3), 45 (+ 8.8), 181 (+

16.8), and 262 (+ 30.1) g fomesafen ha-1 for SS, AR11-LAW-B, C1, and C2 population,

respectively (Figure 2, Table 1). On the basis of LD50 values, AR11-LAW-B, C1, and C2

populations had 3-, 11-, and 16-fold resistance relative to the SS population. The LD50 values of

C1 and C2 populations were relatively similar to the GR50 values. The 0.7X and 1X of the

commercial field dose of fomesafen would kill 50% of the C1 and C2 population, respectively.

Thus, the normal field dose would not control all plants in the C1 and C2 populations, allowing

the resistant plants to proliferate progressively in the next growing season. The PPO-resistant

common waterhemp in Kansas had about the same GR50 value (270 g fomesafen ha-1) as that of

the C2 population18. Although AR11-LAW-B had higher GR50 and LD50 than SS, the

commercial dose of fomesafen can still kill >90% of the AR11-LAW-B population. However,

continued selection had shifted the population response in just two cycles. If each cycle of

selection represents one cropping season, we can predict that resistance to PPO herbicides in the

source field (in 2011) would be apparent in 2013. Unaware of the impending resistance problem

in the field, the farmer happened to have adopted a RoundupReady corn-LibertyLink soybean

140

cropping system, and kept the field clean, except for a few remnant Palmer amaranth in 2015

(1/3 of which, still carried the resistance trait). Thus, prevention of seed production of survivors

cannot be overstated. With good management practices, the evolution of resistant populations

can be slowed down.

3.3 Response of Palmer amaranth population to other foliar-applied herbicides

The AR11-LAW-B population was found to be susceptible to atrazine, dicamba,

glufosinate, glyphosate, and mesotrione but resistant to ALS inhibitors pyrithiobac and

trifloxysulfuron (Table 2). The commercial field dose of pyrithiobac and trifloxysulfuron

controlled AR11-LAW-B 17% and 44%, respectively. The GR50 for pyrithiobac was 3.2 (+ 1.2)

and 44.5 (+ 6.2) g ha-1, respectively, for SS and AR11-LAW-B (Table 3). Similarly, the GR50 for

trifloxysulfuron for SS and AR11-LAW-B was 0.9 (+0.7) and 5.1 (+ 1.1) g ha-1, respectively.

About 0.6X the recommended dose of pyrithiobac and trifloxysulfuron were needed to reduce

the aboveground biomass of AR11-LAW-B by 50%. The AR11-LAW-B population had 14-fold

resistance to pyrithiobac and 5-fold resistance to trifloxysulfuron. The occurrence of multiple

resistances in AR11-LAW-B is not surprising, considering a widespread resistance to ALS

inhibitors among Palmer amaranth in Arkansas28. With the prevalence of glyphosate-resistant

Palmer amaranth population, it is interesting to note that this population is controlled 84% by

glyphosate. The grower of this field had been alternating RoundupReady soybean with rice in the

past years (before 2011), then shifted to LibertyLink soybean in 2011, and used other

management practices for weed control; thus, keeping the population responsive to glyphosate.

This population may or may not be resistant to other PPO herbicides, thus further experiments

need to be conducted to verify its response to other foliar and soil-applied PPO herbicides. A

recent study indicated that soil-applied PPO-inhibitor herbicides remain efficacious in PPO-

141

resistant waterhemp populations in Illinois, although the length of residual activity is shortened

compared with PPO-susceptible waterhemp29. With this population evolving resistance to ALS

and PPO inhibitors, AR11-LAW-B can still be controlled with atrazine, dicamba, glufosinate,

glyphosate, and mesotrione. Reliance on PPO herbicides should be mitigated to hinder or slow

the evolution of PPO resistance in Palmer amaranth. Adequate control of Palmer amaranth can

be achieved with tank-mixes of residual herbicides such as S-metolachlor + metribuzin applied

preemergence followed by pyroxasulfone or S-metolachlor applied early postemergence.

Glufosinate is still an effective option in LibertyLink soybeans; however, it should be coupled

with a preemergence program followed by glufosinate tankmixed with S-metolachlor or

pyroxasulfone early postemergence30. In addition, integration of cultural and mechanical control

practices would be helpful in managing PPO-resistant Palmer amaranth populations31.

3.4 Resistance mechanism in PPO-resistant Palmer amaranth

Results revealed 74 out of 81 C1 plants and all 13 C2 samples tested positive for the

ΔG210 mutation, whereas all the sensitive plants tested negative for the said mutation (data not

shown). This indicated that these survivors were resistant to fomesafen due to target-site

mutation in the PPO gene. Thinglum et al.32 reported that ΔG210 mutation confers resistance to

PPO herbicides in waterhemp populations in Illinois, Kansas, and Missouri suggesting that the

ΔG210 mutation is likely the only known mechanism of resistance to PPO inhibitors in

waterhemp. Palmer amaranth and waterhemp belong to the Amaranthus genus and may,

therefore, have some common morphological, biological, and physiological characteristics and

genomic tendencies. In fact, based on previous DNA sequence comparisons, it was predicted that

the ΔG210 mutation would evolve in Palmer amaranth24. The loss of a glycine residue in the PPO

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gene alters the architecture of the substrate-binding domain of the PPO enzyme33. As a result, the

mutated PPO enzyme has reduced affinity for several PPO-inhibiting herbicides.

Some survivors that did not show the PPO mutation may harbor other resistance

mechanisms such as non-target site-based resistance mechanisms. Diphenyl ether herbicides are

detoxified in soybeans by homoglutathione conjugation34. Similarly, tolerance to the diphenyl

ether fluorodifen in peas is due to rapid conjugation with glutathione35. Alternatively, the lack of

detection of the ΔG210 mutation in some resistant plants could be due to sequence

polymorphisms at the primer binding sites, resulting in false negatives.

3.5 Implications and future research

PPO inhibitors have been used heavily in the past years to combat herbicide-resistant

Palmer amaranth. Before this, PPO herbicides were among the most widely used herbicides for

soybean. As history has shown, intensive use of any herbicides often results in the selection for

genes conferring herbicide resistance in weed populations. This research confirmed the

occurrence of the first Palmer amaranth population to have evolved resistance to a PPO-

inhibiting herbicide. Recurrent selection with the same herbicide significantly increased the

frequency of resistant plants. Because resistance to glyphosate is also rampant, it is expected that

populations with multiple resistance to PPO and ALS inhibitors as well as PPO inhibitors and

glyphosate will evolve. With Palmer amaranth resistant to these modes of action, limited

herbicide options remain for Palmer amaranth control. The evolution of resistance to PPO

inhibitors in Palmer amaranth is a recent phenomenon in the southern US; thus, best

management practices (including diversification of herbicide modes of action) are vital to

manage the spread of resistance, especially in soybean and cotton acres. Since PPO resistance

143

may still be localized, best management practices should be employed on a broader scale

immediately.

Future research will investigate the genetics and mechanism of inheritance to PPO

inhibitors in AR11-LAW-B C1 and C2 population, the efficacy of other PPO herbicides (soil or

foliar) on PPO-resistant populations, the distribution and population genetics of PPO-resistant

Palmer amaranth populations and fitness of multiple-resistant plants.

144

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Table 1. GR50 and LD50 values of original, C1, and C2 AR11-LAW-B populations in Arkansas,

USA.

Population GR50 (g ha-1) R/Sa LD50 (g ha-1) R/Sb

AR11-LAW-B 81.8 (+ 5.1)c 6 44.8 (+ 8.8)c 3

C1 167.8 (+11.9) 13 180.8 (+ 16.8) 11

C2 265.0 (+ 20.4) 21 262.5 (+ 30.1) 16

SSd 12.9 (+ 0.8) 16.3 (+ 1.3)

aResistance levels (R/S) calculated using the GR50 of the resistant population relative to the

susceptible standard. bResistance levels (R/S) calculated using the LD50 of the resistant population relative to the

susceptible standard. cStandard error dHerbicide-susceptible standard population

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Table 2. Response of Amaranthus palmeri AR11-LAW-B population to foliar-applied herbicides

other than protoporphyrinogen oxidase inhibitors, Arkansas, USA.

Herbicide Mortality (%)a Resistance classification

atrazine 100 susceptible

dicamba 100 susceptible

glufosinate 100 susceptible

glyphosate 84 susceptibleb

mesotrione 94 susceptible

pyrithiobac 17 resistant

trifloxysulfuron 44 resistant

aUniform-sized plants (7.5-9 cm tall) were sprayed with atrazine at 2244 g ha-1, dicamba at 280 g

ha-1, glufosinate at 547 g ha-1, glyphosate at 870 g ha-1, mesotrione at 105 g ha-1, pyrithiobac at

73 g ha-1 and trifloxysulfuron at 8 g ha-1. Glufosinate and mesotrione treatments included 3366 g

ammonium sulfate (AMS) ha-1 and 1% crop oil concentrate (COC), respectively. Pyrithiobac and

trifloxysulfuron were applied with 0.25% NIS by volume. Mortality was recorded 21 d after

herbicide application. bPlants were not dead at evaluation time, but survivors incurred high injury and did not grow to

maturity.

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Table 3. GR50 values and resistance levels to ALS inhibitors in AR11-LAW-B Palmer amaranth

population in Arkansas, USA.

Population Pyrithiobac Trifloxysulfuron

GR50 (g ha-1) R/Sa GR50 (g ha-1) R/S

AR11-LAW-B 44.5 (+6.2)b 14 5.1 (+1.1) 5

SSc 3.2 (+1.2) - 0.9 (+0.7) -

aResistance levels (R/S) calculated using the GR50 of the resistant population relative to the

susceptible standard. bStandard error of estimate cHerbicide-susceptible standard population

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Figure 1. Shoot biomass reduction (%) of PPO-resistant and –susceptible Palmer amaranth

population, 21 d after fomesafen treatment.Treatment means (n = 10) + 1 standard error are

plotted and fitted to a regression curve. Data were best described with a nonlinear, sigmoidal,

three-parameter Gompertz regression function, y =a*exp{-exp[-b*(x-c0]}.

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Figure 2. Dose-response curves of PPO-resistant and –susceptible Palmer amaranth population

from Arkansas.Mortality (%) were obtained at 21 d after herbicide treatment. Treatment means

(n = 10) + 1 standard error are plotted and fitted to a regression curve. Data were best described

with a nonlinear, sigmoidal, three-parameter Gompertz regression function, y =a*exp{-exp[-

b*(x-c0]}.

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CHAPTER VI

FREQUENCY OF GLY-210 DELETION MUTATION AMONG

PROTOPORPHYRINOGEN OXIDASE INHIBITOR-RESISTANT PALMER

AMARANTH (AMARANTHUS PALMERI) POPULATIONS

© Weed Science Society of America, 2017

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Abstract

The widespread occurrence of Palmer amaranth resistant to acetolactate synthase

inhibitors and/or glyphosate led to the increased use of protoporphyrinogen oxidase (PPO)-

inhibiting herbicides. This research aimed to: (1) evaluate the efficacy of foliar-applied

fomesafen to Palmer amaranth, (2) evaluate cross-resistance to foliar PPO inhibitors and efficacy

of foliar herbicides with different mechanisms of action, (3) survey the occurrence of the PPO

Gly-210 deletion mutation among PPO inhibitor– resistant Palmer amaranth, (4) identify other

PPO target-site mutations in resistant individuals, and (5) determine the resistance level in

resistant accessions with or without the PPO Gly-210 deletion. Seedlings were sprayed with

fomesafen (263 g ai ha−1), dicamba (280 g ai ha−1), glyphosate (870 g ai ha−1), glufosinate (549 g

ai ha−1), and trifloxysulfuron (7.84 g ai ha−1). Selected fomesafen-resistant accessions were

sprayed with other foliar-applied PPO herbicides. Mortality and injury were evaluated 21d after

treatment (DAT). The PPX2L gene of resistant and susceptible plants from a selected accession

was sequenced. The majority (70%) of samples from putative PPO-resistant populations in 2015

were confirmed resistant to foliar-applied fomesafen. The efficacy of other foliar PPO herbicides

on fomesafen-resistant accessions was saflufenacil>acifluorfen = flumioxazin>carfentrazone =

lactofen >pyraflufen-ethyl>fomesafen>fluthiacet-methyl. With small seedlings, cross-resistance

occurred with all foliar-applied PPO herbicides except saflufenacil (i.e., 25% with acifluorfen,

42% with flumioxazin). Thirty-two PPO-resistant accessions were multiple resistant to

glyphosate and trifloxysulfuron. Resistance to PPO herbicides in Palmer amaranth occurred in at

least 13 counties in Arkansas. Of 316 fomesafen survivors tested, 55% carried the PPO Gly-210

deletion reported previously in common waterhemp. The PPO gene (PPX2L) in one accession

(15CRI-B), which did not encode the Gly-210 deletion, encoded an Arg-128-Gly substitution.

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The 50% growth reduction values for fomesafen in accessions with Gly-210 deletion were 8- to

15-fold higher than that of a susceptible population, and 3- to 10-fold higher in accessions

without the Gly-210 deletion.

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Introduction

Palmer amaranth is one of the most troublesome and economically damaging agronomic

weeds in the southern United States. It is able to adapt to diverse climatic and agricultural

conditions. The photosynthetic rate of Palmer amaranth (81µmol m−2s−1) is three to four times

that of corn (Zea mays L.), cotton (Gossypium hirsutum L.), and soybean [Glycine max (L.)

Merr.] (Ehleringer 1983). This high photosynthetic rate translates into its rapid growth rate of

5cm day−1 under optimum growing conditions (Horak and Loughin 2000). Consequently, this

rapid growth rate results in a narrow window of opportunity for effective POST herbicide

application. Palmer amaranth control becomes difficult when it is >10-cm tall (Riar et al. 2013).

A female Palmer amaranth can produce up to 1 million seeds; however, its seeds are relatively

short-lived in the soil, with about 80% mortality in 3yr (Sosnoskie et al. 2014). With its fast

growth rate, high fecundity, season-long emergence, high photosynthetic rate, and high

propensity to evolve herbicide resistance, Palmer amaranth has become a serious weed in row

crops and vegetables (Ehleringer et al.1997; Guo and Al-Khatib 2003; Jha and Norsworthy 2012;

Steckel 2007). Palmer amaranth infestation can reduce corn, cotton, and soybean yield 91%,

77%, and 78%, respectively (Bensch et al. 2003; Fast et al. 2009; Massinga et al. 2001). In the

past decade, reports abound of Palmer amaranth evolving resistance to glyphosate and other

herbicides (Culpepper et al. 2006; Jha et al. 2008; Norsworthy et al. 2008). To date, Palmer

amaranth has been confirmed resistant to acetolactate synthase (ALS) inhibitors (Burgos et al.

2001; Horak and Peterson 1995), dinitroanilines (Gossett et al. 1992), 4-hydroxyphenylpyruvate

dioxygenase (HPPD) inhibitors (Jhala et al. 2014), glyphosate (Culpepper et al. 2006;

Norsworthy et al. 2008), photosystem II herbicides (Vencill et al. 2011), and most recently,

protoporphyrinogen oxidase (PPO) inhibitors (Salas et al. 2016).

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Several chemical families inhibit PPO activity, including diphenylethers (e.g.,

acifluorfen, fomesafen, lactofen, oxyfluorfen), N-phenylphthalimide (e.g., flumioxazin,

flumiclorac), phenylpyrazoles (e.g., fluazolate, pyraflufen-ethyl), oxadiazole (e.g., oxadiazon),

oxazolidinones (e.g., pentoxazone), pyrimidinediones (e.g., saflufenacil), thiadiazole (e.g.,

fluthiacet-methyl), and triazolinone (e.g., carfentrazone, sulfentrazone) (Heap2017).

Diphenylether herbicides are used PRE or POST. Some triazolinones and N-phenylphthalimide

have high soil activity and are phytotoxic to the crop when applied foliar, hence most are

commonly used PRE (Dayan and Duke 2010). There are two known nuclear PPO genes in

plants, PPX1 and PPX2, which encode plastid- and mitochondrial-targeted PPO isoforms,

respectively; however, some PPX2 isoforms are dual targeted to both organelles (Lermontova

and Grimm 2000; Lermontova et al. 1997; Watanabe et al. 2001). The inhibition of PPO causes

accumulation of protophorphyrinogen IX, which leaks from the plastid to the cytoplasm, where it

is oxidized rapidly into photosensitive protoporphyrin IX (Becerril and Duke 1989; Jacobs et al.

1991; Lee et al. 1993). Upon exposure to light, protoporphyrin IX generates singlet oxygen

molecules that cause lipid peroxidation, membrane destruction, and ultimately cell death

(Becerril and Duke 1989; Jacobs et al. 1991). The first weed species to evolve resistance to PPO-

inhibiting herbicides was common waterhemp in 2001 (Shoup et al. 2003). Resistance to PPO

inhibiting herbicides in Palmer amaranth was first reported in Arkansas and is confirmed also in

Tennessee and Illinois (Heap 2017; Salas et al. 2016; L Steckel and K Gage, personal

communication). When this research was conducted, resistance to PPO herbicides in Amaranthus

spp. was attributed to a target-site mutation in the PPX2 gene only (Patzoldt et al. 2006; Salas et

al. 2016). The Gly-210 deletion mutation was present in all PPO-resistant waterhemp

populations in Illinois, Kansas, and Missouri (Thinglum et al. 2011). However, a novel PPO

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mutation, Arg-98-Leu, was detected in PPO-resistant common ragweed (Ambrosia artemisiifolia

L.) (Rousonelos et al. 2012).

This study was conducted to investigate the response of Palmer amaranth from Arkansas

to various foliar-applied PPO-inhibiting herbicides (acifluorfen, carfentrazone, flumioxazin,

fluthiacetmethyl, fomesafen, pyraflufen-ethyl, saflufenacil) and to foliar-applied herbicides with

different modes of action (dicamba, glyphosate, glufosinate, trifloxysulfuron). The study also

aimed to determine the frequency of Gly-210 deletion among the PPO-resistant biotypes,

identify other PPO target-site mutations that endow resistance to PPO-inhibiting herbicides, and

compare the resistance level to fomesafen in accessions that contained or lacked the Gly-210

deletion.

Materials and Methods

Plant Materials. A total of 124 Palmer amaranth accessions were collected in the summer

between 2008 and 2015 from 23 counties in Arkansas, primarily from the eastern half of the

state. Sampling sites were identified by county extension agents and crop consultants. Samples in

2008 and 2009 were collected from 18 counties (17 from eastern Arkansas and 1 from central

Arkansas) to survey resistance to glyphosate. Seven counties were sampled in 2011 from fields

that were planted with LibertyLink® crops to verify differential response to glufosinate. Fifty-

two fields from 16 counties were sampled in 2014. Samples in 2015 (23 accessions) were

collected specifically to survey fields with remnant Palmer amaranth after having been sprayed

with PPO herbicides. Inflorescences from at least 10 mature female plants−1 field were

collected, dried, and threshed. Equal amounts of seeds from each plant were mixed to make a

composite seed sample to represent the field. A sensitive standard population (SS) was included

in each experiment for comparison. Plants were grown in a greenhouse maintained at 32/25±3°C

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day/night temperature with a 16:8-h day:night regime and photon flux density of 0.0005 mol m−2

s−1. Plants were planted using commercial potting soil (Sunshine® Premix No. 1; Sun Gro

Horticulture, Bellevue, WA), watered daily, and fertilized with Miracle-Gro® (Scotts Miracle-

Gro, Marysville, OH) every 2 wk.

Palmer Amaranth Response to Foliar-applied Fomesafen. One hundred-twenty four Palmer

amaranth accessions (Table 1) were tested in the greenhouse for resistance to fomesafen.

Composite seed samples were planted in 24 by 54 cm cellular trays. Seedlings were thinned to 1

plant cell−1 and sprayed with 263g ai ha−1 fomesafen (Flexstar®, Syngenta Crop Protection,

Greensboro, NC 27419) when seedlings were 7- to 8-cm tall. The herbicide was applied with

0.5% by volume nonionic surfactant (NIS) (Induce®, Helena Chemical, Collierville, TN 38017)

using a laboratory sprayer equipped with a flat-fan spray nozzle delivering 187 L ha−1 at 221

kPa. The experiment was conducted in a randomized complete block design with two

replications and repeated. Each replication consisted of 50 seedlings grown in a cellular tray at 1

seedling cell−1. The plants were assessed visually relative to nontreated plants at 21 d after

treatment (DAT) using an injury scale of 0 to 100, where 0% = no visible injury and 100% =

complete desiccation. Survivors with 0% to 10%, 11% to 30%, 31% to 60%, and 61% to 89%

injury were classified as highly resistant, resistant, moderately resistant, and slightly resistant,

respectively. Individuals with 90% injury or higher were considered sensitive. In this

experiment, an accession with >10% survivors that were at least slightly resistant was considered

to be PPO resistant. Data were analyzed using hierarchal clustering in JMP Pro v. 13 (SAS

Institute, Cary, NC.).

Response to Other Foliar-applied PPO Herbicides. Twelve accessions that had low mortality

(<83%) from foliar-applied fomesafen were bioassayed in the greenhouse for their response to

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other foliar-applied PPO herbicides. A sensitive standard accession was also included. Seedlings

(100 per accession, 10-cm tall) were treated with the recommended doses of acifluorfen,

carfentrazone, flumioxazin, fluthiacet-methyl, lactofen, pyraflufenethyl, or saflufenacil (Table 2).

Carfentrazone, pyraflufen-ethyl, flumioxazin, and fluthiacet-methyl treatments included 0.25%

NIS (v/v), whereas acifluorfen included 0.125% NIS (v/v). Lactofen was sprayed with 0.5% crop

oil concentrate (v/v). Saflufenacil was sprayed with 1% methylated seed oil and 1% ammonium

sulfate (w/v). Following herbicide applications, the plants were placed in the greenhouse,

grouped by herbicide, and the accessions were randomized within each herbicide group.

Mortality and injury of survivors were evaluated at 21 DAT. In the second run of the experiment,

herbicides were sprayed when seedlings were 5- to 8-cm tall. Data were analyzed, by herbicide,

using JMP Pro v. 13 (SAS Institute, Cary, NC).

Response to Foliar-applied Non-PPO Herbicides. Seventy-three accessions collected in 2014

and 2015 were tested in the greenhouse for response to dicamba, glufosinate, glyphosate, and

trifloxysulfuron (Table 2). The SS accession was also included for comparison. The experiment

was conducted in a completely randomized design with two replications and two runs. Each

replication consisted of 50 plants grown in a cellular tray. Composite seed samples were planted

as described earlier. The foliar herbicides were applied to uniform-sized plants (7.6-cm tall).

Glufosinate and trifloxysulfuron were sprayed with 3,366g AMS ha−1 and 0.25% NIS (v/v),

respectively. Herbicide applications were made as described earlier. Plants were evaluated for

injury and mortality at 21 DAT. Data were analyzed by herbicide using JMP Pro v. 13 (SAS

Institute, Cary, NC). Response to glyphosate was analyzed using hierarchal clustering,

collectively considering mortality and injury of survivors.

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Mechanism of Resistance to PPO Herbicides. A total of 316 survivors from 47 accessions

treated with foliar-applied fomesafen were analyzed for the Gly-210 deletion mutation (Patzoldt

et al. 2006; Salas et al. 2016). Young leaf tissues were collected from up to 10 survivors each of

35 accessions sprayed with the recommended dose of foliar-applied fomesafen in the greenhouse

bioassays. In addition, leaf tissues were collected from 82 plants in 14 fields with high

population density of Palmer amaranth after having been sprayed with PPO inhibitors, among

other herbicides. DNA was extracted using a modified Cetyltrimethylammonium bromide

(CTAB) protocol (Sales et al. 2008) and quantified using a NanoDrop spectrophotometer (ND-

1000, Thermo Scientific, Wilmington, DE). An allelespecific PCR assay was used to detect the

codon Gly210 following the protocol described for common waterhemp, which also worked for

Palmer amaranth (Lee et al. 2008; Salas et al. 2016). Some PPO-resistant plants that did not have

the Gly-210 deletion mutation were selected for sequencing of the PPX2L gene. Total RNA was

extracted using the RNeasy extraction kit (Qiagen 74903, Valencia, CA) and converted to cDNA

using the Reverse Transcription kit (Promega A3500, Madison, WI). Initially a partial sequence

of the PPX2L gene was amplified using the primer pair ppx2-1F (5′-

GTAATTCAATCCATTACCCACCTT-3′) and ppx2-3R (5′-TTACGCGGTCTTCTCATCCAT-

3′) and sequenced using the internal primers: ppx2-1R (5′-TTCCATACGTCGGGAAATGT-3′),

ppx2-2F (5′-TGTTGGAACCATTTCTCTGG-3′), ppx2-2R (5′-

GGGGATAAGAACTCCGAAGC-3′) and ppx2-3F (5′-GATGCTGTGGTTGTCACTGC-3′).

Eventually, the full-length sequences of PPX2L in PPO-sensitive and PPO-resistant plants were

obtained (GenBank accession nos. MF583744 and MF583746) by designing primers based on

the upstream and downstream gene sequence of Prince-of-Wales feather (Amaranthus

hypochondriacus L.) (GenBank accession no. EU024569.1). These additional primers were:

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ppx2-5′UTR (5′-TGGCAGATTGAGACAAAATT GG-3′) and ppx2-3 ′UTR (5′-

GGCAGAAAAGTCAC TGCACA-3′).

Fomesafen Dose–Response Bioassay. Seven PPO-resistant accessions, including the SS, were

used in whole-plant bioassays to determine the resistance level to fomesafen. Three accessions

that contained the Gly-210 deletion (14MIS-H, 15MIS-C, 15MIS-E) and four accessions that

lacked the Gly-210 deletion mutation (14CRI-C, 15CRI-B, 15PHI-A) were selected. Seedlings

were grown in 15-cm-diameter pots and thinned to 5 plants pot−1. Seedlings, 5- to 7-cm tall, were

sprayed with 10 doses of fomesafen from 0 to 2,107g ai ha−1. The SS was sprayed with 9 doses

from 2 to 263 g ha −1, corresponding to 1/128 to 1X the recommended dose. A nontreated check

was included. The herbicide was applied with 0.5% NIS. The experiment was conducted in a

randomized complete block design with four replications. At 21 DAT, plants were cut at the soil

surface, shoots were dried for 48 h at 60°C, and the dry weights were recorded. Data were

analyzed using SAS JMP Pro v.13 (SAS Institute, Cary, NC) in conjunction with SigmaPlot v.13

(Systat Software, San Jose, CA) for regression analysis. The percentage biomass reduction was

fit to a nonlinear, sigmoid, four-parameter logistic regression model defined by

𝑦 = 𝑐 + [(𝑑 − 𝑐)/(1 + 𝑒{−𝑎(𝑥 − 𝑏)})]

where y is the biomass reduction expressed as percentage relative to the untreated control, a is

the growth rate, b is the inflection point, c is the lower asymptote, d is the upper asymptote, and x

is the fomesafen dose. Estimates of herbicide dose that cause 50% growth reduction (GR50) were

determined using the fitted regression equation.

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Results and Discussion

Palmer Amaranth Response to Foliar-applied Fomesafen. The frequency distribution of

mortality from fomesafen treatment was highly skewed, with a greater proportion of high

mortality; therefore, the median values were used to describe the data, except for the 2015

accessions (Figure 1). The 2008 accessions generally incurred 97% mortality with the field dose

of fomesafen, except for three accessions with <90% mortality. One rare accession from Phillips

County had only 74% mortality; however, the survivors incurred >75% injury and did not

produce seeds. Thus, the early samples had individuals that were relatively more tolerant to

fomesafen than others. The accessions in 2009 and 2011 were all susceptible, but one of two

accessions with 95% mortality, 11LAW-B, contained rare individuals carrying the Gly-210

deletion mutation (Salas et al. 2016). Eleven accessions (22%) collected in 2014 and 16

accessions (70%) in 2015 were classified resistant. The 2015 samples were from fields with

suspected resistance to PPO herbicides, and the results confirmed most growers’ suspicions. The

mortality data with fomesafen treatment was normally distributed from 23% to 100%, with an

average of 71%. Accessions classified as PPO resistant in 2015 consisted of individuals with

variable response to fomesafen (Figure 2). For example, 15CLA-A had 50% survivors and

almost all showed ≤10% injury. On the other hand, 15GRE-A had 75% survivors, and only about

one-half showed ≤10% injury. Overall in 2015, 16 accessions from 10 counties showed mostly

<90% mortality with fomesafen and were classified resistant based on further evaluations

discussed in succeeding sections. This shows that resistance to PPO herbicides among Palmer

amaranth has evolved quasi-simultaneously across counties in Arkansas.

Prior to 2015, there were fields with a few Palmer amaranth remaining after PPO

herbicide application, but such low numbers did not catch the growers’ attention because of the

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expected variability in plant response under various environmental conditions. In some cases,

any escapes would have been blamed on various factors. Continued selection with different

herbicides, but with the same PPO-inhibitor mode of action, has increased the frequency of

surviving individuals to a field-scale, observable level. PPO-inhibiting herbicides are used

extensively to control glyphosate- and ALS-resistant Palmer amaranth in soybean and cotton

(Salas et al. 2016). Prior to the widespread glyphosate-resistance problem in Amaranthus,

fomesafen had been used primarily as PRE or POST herbicide for broadleaf weed control in

soybean. Since fomesafen commercialization in the 1960s, several other herbicides targeting the

PPO enzyme have been commercialized. With the expansion of PPO-herbicide use pattern to

cotton and PPO-herbicide use in preplant applications, the selection pressure from this mode-of-

action group has intensified greatly. Considering that rare PPO-resistant Palmer amaranth

individuals were detected retroactively in a population collected in 2011, it took 4 yr before

several reports arose of field-level escapes from PPO herbicide application in 2015.

Fields sampled in 2015 were those with remnant infestations of Palmer amaranth after a

weed management program that included multiple applications of a PPO herbicide. Most of

those fields had plants resistant to 263 g ha−1 fomesafen. The frequency of resistance reported

here pertained mostly to fields with putative PPO herbicide– resistance problem. A random

survey of fields infested with Palmer amaranth across the state, irrespective of cropping history,

is expected to produce a lower frequency of resistance to PPO herbicides. Of the 23 fields

verified for resistance in 2015, only two had 100% mortality with a field dose of fomesafen. The

rest had resistant individuals showing different levels of injury of survivors (Figure 2). The

majority of survivors from three accessions (15CLA-A and 15CRI-A) incurred minimal injury

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(0% to 10%). High frequency of PPO-resistant individuals among the 2015 accessions threatens

the continued use of PPO herbicides.

Multivariate cluster analysis was conducted to group the 2014 and 2015 accessions based

on their response to the field use rate of fomesafen, taking into account both mortality data and

injury of survivors. The 2014 and 2015 accessions differentiated into four clusters (Figure 3).

The 50 accessions belonging to the first cluster were the most sensitive, the majority of which

were from the quasi-random collection in 2014. The second cluster is composed of 19 resistant

accessions with the majority of survivors showing >60% injury, from the 2014 and 2015

collection. The third cluster comprises two resistant accessions in which most survivors incurred

low (11% to 30%) injury. The fourth cluster contains the three resistant accessions in which a

large number of survivors incurred the lowest (<11%) injury. This indicates that a large number

of PPO-resistant plants incurred low to moderate levels of injury from field use rate of

fomesafen, allowing them to mature and reproduce. The evolution of observable, population-

level resistance to PPO herbicides in multiple fields in 2015 demonstrated the quasi-

simultaneous evolution of resistance to PPO herbicides in Palmer amaranth. This occurred after

several decades of use primarily in soybean, then more recently in cotton. The proportion of

resistant populations that evolved via gene flow (pollen or seed) or via independent selection is

yet to be determined.

Response to Other Foliar-applied PPO-inhibiting Herbicides. The response of Palmer

amaranth to other foliar-applied PPO herbicides and to other non-PPO foliar-applied herbicides

was evaluated, because PPO herbicides are used heavily to control ALS- and glyphosate-

resistant Palmer amaranth. Twelve of the most fomesafen-resistant accessions were tested with

other PPO-inhibiting herbicides (Table 3). Mortality with pyraflufen-ethyl and fluthiacet-methyl

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was similar across runs; data were therefore combined. These herbicides controlled the SS 100%.

The mortality of all accessions, excluding the SS, was <72% with fluthiacet-methyl. Palmer

amaranth generally has variable sensitivity to this PPO herbicide and is therefore not listed on

the label; whereas this herbicide is expected to provide only partial control of common

waterhemp (Anonymous 2011). The mortality rating of all PPOresistant accessions was at least

17% lower than the SS when treated with pyraflufen-ethyl, with the exception of 14CRI-C

(>90% mortality). Pyraflufen-ethyl also is naturally weak on Palmer amaranth, although it killed

the SS in these experiments, as did the other herbicides.

Mortality was different between runs for acifluorfen, carfentrazone, flumioxazin,

lactofen, and saflufencil; data were therefore analyzed separately. In general, mortality was

higher in the second run than in the first run due to smaller plant size at the time of herbicide

treatment. With acifluorfen, only 2 of 12 fomesafen-resistant accessions tested (14CRI-C and

15CRI-C) were effectively controlled (>90% mortality) in the first run; however, 75% of

fomesafen-R accessions were susceptible to acifluorfen when smaller plants were sprayed in the

second run. With carfentrazone, all 12 fomesafen-resistant accessions showed only 48% to 83%

mortality in the first run. Similar results were observed in the second run. With flumioxazin,

eight accessions that had ≥74% mortality in the first run were verified to be sensitive in the

second run. About 42% of fomesafen-resistant accessions tested were cross-resistant to foliar-

applied flumioxazin. The mortality of all accessions was <70% with lactofen in the first run, with

the exception of 15CRI-C (93% mortality). In the second run, all accessions were still poorly to

moderately controlled, except for 14CRI-C and 15CRI-C (>90% mortality). Saflufenacil was

effective only on five accessions (>90% mortality) with larger seedlings. With smaller seedlings,

all 12 fomesafen-resistant accessions showed >97% mortality with saflufenacil. Across all

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chemistries, regardless of herbicidal strength, application on small seedlings is necessary for

maximum possible efficacy. Application on bigger seedlings is risky. Previous reports indicated

that resistance of common waterhemp to foliar-applied PPO herbicides becomes prevalent at the

4- to 6-leaf stage (Falk et al. 2006). Plant size is a critical factor in the efficacy and consistency

of performance of most foliar-applied herbicides. When growers miss the application window,

selection for resistance is expected to be stronger. If survival in greenhouse bioassays is seen to

be consistent across replications and repetitions, then the risk of having survivors in the field

populations is high.

Consistent in both runs, fluthiacet-methyl was the least effective and saflufenacil was the

most effective of all foliar-applied PPO herbicides. The response to PPO herbicides, in order of

decreasing efficacy, was as follows: saflufenacil>acifluorfen = flumioxazin> carfentrazone =

lactofen>pyraflufen-ethyl>fluthiacetmethyl. In this series, fomesafen would fall between the last

two herbicides. The fomesafen-resistant accessions were generally cross-resistant to other foliar-

applied PPO herbicides, with the exception of saflufenacil. However, saflufenacil is not an in-

crop option for soybean or cotton. Although saflufenacil showed the greatest efficacy, some

accessions already showed reduced sensitivity to the field use rate of saflufenacil when applied

to 10-cm-tall seedlings. Previous studies on diphenylether-resistant tall waterhemp [Amaranthus

tuberculatus (Moq.) Sauer] also showed cross-resistance to other foliar-applied PPO herbicide

families (Patzoldt et al. 2005; Shoup et al. 2003; Wuerffel et al. 2015a).

Response to Foliar-applied Non–PPO Inhibiting Herbicides. Palmer amaranth accessions

collected between 2014 and 2015 were tested with dicamba, glufosinate, glyphosate, and

trifloxysulfuron. The 73 accessions, including the SS accession, differentiated into four clusters

in response to glyphosate (Figure 4A). The first cluster was composed of 13 resistant accessions

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that had 0% to 36% mortality with glyphosate, with the majority of survivors incurring 31% to

60% injury. Twenty-three accessions constituted the second cluster, with resistance to glyphosate

in which a large number of the survivors incurred <11%injury. The third cluster was composed

of 24 accessions with ≥65% mortality, 17 of which were classified as sensitive (at least 90%

mortality). The fourth cluster included 14 resistant accessions (36% to 68% mortality), in which

most of the survivors sustained >60% injury from glyphosate treatment. Overall, about 50% of

accessions (Clusters 1 and 2) were resistant to glyphosate, with mortality ranging from 0%

to42%. In addition, all accessions were also resistant to trifloxysulfuron, showing <86%

mortality (Figure 4B). The widespread occurrence of ALS- and glyphosate-resistant Palmer

amaranth in Arkansas was reported previously (Burgos et al. 2009). The ALS-inhibiting

herbicides and glyphosate had been used extensively in the past. Overall, the 27 PPO-resistant

accessions were also resistant to either ALS inhibitor or glyphosate, or both (Figure 5; Table 4).

The majority of PP-Oresistant accessions (85%) exhibited resistance to both glyphosate and

trifloxysulfuron.

The 2014 and 2015 accessions were all sensitive to the 549g ai ha−1 glufosinate (>90%

mortality) (Figure 4B). All accessions incurred >95% mortality, except for 15CRI-B (91%). In

the same manner, dicamba controlled all accessions (Figure 4B). Dicamba caused >90%

mortality in 61% of the accessions. Mortality of the remaining accessions was <89%; however,

the survivors incurred >75% injury. Anecdotal reports, however, indicated poor performance of

dicamba in fields infested with PPO-resistant Palmer amaranth.

Prevalence of the PPO-Gly-210 Deletion Mutation in Resistant Palmer Amaranth. The Gly-

210 deletion in the PPX2L gene confers resistance to PPO-inhibiting herbicides in PPO-resistant

common waterhemp and Palmer amaranth (Patzoldt et al. 2006; Salas et al. 2016; Wuerffel et al.

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2015b). A point mutation in PPX2 of common ragweed also confers resistance to PPO

herbicides; however, whereas a plastid-targeting signal was not found to be encoded by PPX2 in

common ragweed (Rousonelos et al. 2012), PPX2L in common waterhemp encodes plastidic and

mitochondrial isoforms of PPO (Patzoldt et al. 2006). This dual-targeting phenomenon has been

reported previously for spinach (Spinacia oleracea L.) and corn (Watanabe et al. 2001). The

Gly-210 deletion in imparts herbicide resistance to the dualtargeted protein by altering the

architecture of the substrate binding domain (Dayan et al. 2010).

The molecular survey was carried out on 316 fomesafen-resistant plants from 38

accessions representing 13 counties in Arkansas including Clay, Conway, Crittenden, Greene,

Independence, Jackson, Lawrence, Lee, Independence, Mississippi, Phillips, White, and

Woodruff (Figure 6). The Gly-210 codon deletion mutation was found in 46% and 60% of the

survivors from 2014 and 2015 accessions, respectively (unpublished data). The PPO Gly-210

was prevalent among the PPO-resistant accessions; however, a substantial number of the

resistant plants did not carry this mutation (Figure 7, A and B). In most of the PPO-resistant

accessions (68%), resistant individuals were mixtures of carriers and noncarriers of the Gly-210

deletion. Only 13% of the PPO-resistant accessions (n = 6) contained individuals that were all

carriers of the Gly-210 deletion. In nine accessions, none of the resistant individuals carried the

Gly-210 deletion. This indicates that either an alternative target-site mutation is present or

another resistance mechanism is occurring within and among resistant populations in the field.

To verify the occurrence of alternative target-site mutations, we generated a 1,542-bp sequence

of PPX2L in selected resistant individuals without the Gly-210 deletion (GenBank accession no.

MF583745) from the noncarrier accession 15CRI-B. Resistant plants in this accession contained

a different amino acid mutation, Arg-128-Gly. This mutation was reported recently in parallel

169

investigations of PPO-resistant Palmer amaranth, along with an Arg-128-Met mutation

(Giacomini et al. 2017). A mutation at the homologous site was identified previously in PPO-

resistant common ragweed in the form of an Arg-98-Leu substitution (Rousonelos et al. 2012).

Our survey of more than 300 PPO inhibitor– resistant individuals representing 38 field

populations showed that a resistant population may contain a mixture of plants carrying either

one of these mutations. Henceforth, testing for these mutations should be done simultaneously on

suspected PPO inhibitor–resistant plants using available tools (Giacomini et al. 2017).

Sequencing of Palmer amaranth with different resistance levels to PPO inhibitors, with or

without the Gly-210 mutation, is ongoing.

The occurrence of other resistance-conferring mutations in other loci of the PPO gene is

rare, as indicated by previous research. Many single or double point mutations were reported in

mutant PPO genes in an attempt to obtain PPO herbicide– resistant Arabidopsis (Li and Nicholl

2005). Those authors’ data showed that single point mutations either provided low resistance or

resulted in substantial fitness penalty.

Resistance Level to Fomesafen. The fomesafen dose that caused 50% growth reduction (GR50)

ranged from 116 to 232g ha−1 in accessions that contained the Gly-210 deletion, whereas GR50

ranged from 51 to 153g ha−1 in accessions that lacked the Gly-210 deletion (Table 5). Based on

these GR50 values, the level of resistance to fomesafen relative to the SS ranged from 8- to 15-

fold in accessions that contained the Gly-210 and from 3- to 10-fold in accessions that lacked the

Gly-210 deletion. The dose–response bioassay indicated high resistance level in accessions

carrying the Gly-210 deletion mutation. However, some accessions (14CRI-G and 15CRI-B) that

lacked the Gly-210 deletion had GR50 values comparable to those of accessions that contained

the Gly-210 deletion. Accession 15CRI-B, which contained the Arg-128-Gly mutation, had

170

similar GR50 values to those of the resistant accessions carrying the Gly-210 deletion. This

indicates that Gly-210 and Arg-128-Gly mutations result in comparable levels of resistance to

fomesafen. Accessions 14CRI-C and 15PHI-A, which lacked the Gly-210 deletion, had lower

GR50 values than the other PPO-resistant accessions but higher GR50 values than the SS. These

accessions have not yet been studied for the occurrence of other PPO mutations or other

resistance mechanisms. Although our results indicated that the Arg-128-Gly mutation may

confer a resistance level similar to that of the Gly-210 deletion, additional data are needed to

quantify the impact of this mutation on resistance to PPO herbicides. In addition, we have not

ruled out the possibility that other resistance mechanism (s) exist in some PPO inhibitor–resistant

accessions, particularly those exhibiting low-level resistance to PPO inhibitors. It is also possible

also the populations showing high-level resistance harbor multiple resistance mechanisms, as in

the case of ALS-resistant turnipweed [Rapistrum rugosum (L.) All.] (Hatami et al. 2016).

The increasing number of Palmer amaranth populations with resistance to PPO herbicides

is a great concern, because this limits herbicide options for cotton and soybean. Most PPO-

resistant populations were also resistant to glyphosate and trifloxysulfuron, which leaves almost

no herbicides for POST Palmer amaranth control. The remaining POST herbicide options for

PPO-resistant Palmer amaranth include glufosinate in LibertyLink® crops, dicamba in Roundup

Xtend® or Engenia®, or 2,4-D in Enlist Duo® crops. However, overdependence on glufosinate

and phenoxy herbicides must be avoided, as some Palmer amaranth populations show high

tolerance to dicamba (unpublished data). If we lose glufosinate and dicamba, there will be zero

POST options for weed control in soybean and cotton, unless HPPD and 2,4-D traits are

commercialized soon.

171

Overall, the majority of the PPO-resistant individuals carried the PPO Gly-210 deletion.

An alternative target-site mutation, Arg-128-Gly, was identified in resistant plants of one

accession analyzed that did not carry the Gly-210 mutation. The occurrence of other resistance-

conferring mutations or other resistance mechanisms is being investigated. The combination of

these mutations in one plant may be lethal, but the presence of these mutations in different plants

in one field, or different mutations in proximal fields, may accelerate the evolution and spread of

resistance to PPO inhibitors in Palmer amaranth.

172

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175

Table 1. Number of Palmer amaranth accessions in

Arkansas, USA tested with foliar-applied fomesafen

at 263 g ha-1.

a Palmer amaranth samples were collected from

fields with history of glyphosate, glufosinate, or

PPO-inhibiting herbicide use. Sampling in 2015

was done specifically to survey fields with remnant

Palmer amaranth population after having been

sprayed with PPO herbicides.

Year collecteda Number of accessions

2008 25

2009 10

2011 16

2014 50

2015 23

Total 124

176

17

6

Table 2. Common, trade names, and manufacturers of herbicides used in the study.

Site of

action

Application Common name Trade Rate Company Company

website

g ai ha-1

PPO foliar acifluorfen Ultra Blazer 2SL 560 United Phosphorus, Inc.,

King of Prussia, PA 19406

www.upi-

usa.com

PPO foliar carfentrazone Aim 2EC 280 FMC Corporation,

Philadelphia, PA 19103

www.fmc.com

PPO foliar flumioxazin Valor SX 51 WDG 70.6 Valent USA Corporation,

Walnut Creek, CA

www.valent.com

PPO foliar fluthiacet-methyl Cadet 0.91EC 0.672 FMC Corporation,


www.fmc.com

PPO foliar lactofen Cobra 2EC 224 Valent USA Corporation,

Walnut Creek, CA

www.valent.com

PPO foliar pyraflufen-ethyl ET 0.2EC 3.64 Nichino America,

Wilmington, DE 19808

www.nichino.net

PPO foliar saflufenacil Sharpen 4F 24.6 FMC Corporation,


www.fmc.com

PPO soil fomesafen Reflex 2SL 280 Syngenta Crop Protection,

LLC, Greensboro, NC

27419

www.syngenta-

us.com

PPO soil flumioxazin Valor SX 51 WDG 70.6 Valent USA Corporation,

Walnut Creek, CA

www.valent.com

PPO soil saflufenacil Sharpen 4F 49.3 FMC Corporation,


www.fmc.com

PPO soil sulfentrazone Spartan 4F 280 FMC Corporation,


www.fmc.com

177

17

6

Table 2 (Cont.)

Site of

action

Application Common name Trade Rate Company Company

website

g ai ha-

1

PPO soil oxyfluorfen GoalTender 4E 280 Dow AgroSciences LLC,

Indianapolis, IN 46268

www.dowagro.c

om

Auxin foliar dicamba Clarity 4SL 280a BASF Corporation,

Research Triangle Park, NC

27709

https://www.basf

.com

Glutamine

synthetase

foliar glufosinate Liberty 280SL 549 Bayer CropScience LP,

Research Triangle Park, NC

27709

www.bayer.com

EPSPS foliar glyphosate Roundup PowerMAX

4.5SL

870a Monsanto Company, St.

Louis, MO 63167

www.monsanto.

com

ALS foliar trifloxysulfuron Envoke 75 7.84 Syngenta Crop Protection,

LLC, Greensboro, NC

27419

www.syngenta-

us.com

a g ae ha-1

177

178

178

Table 3. Response of fomesafen-resistant Palmer amaranth accessions to the recommended rate of various foliar-applied PPO

herbicides.

Mortalityb

Accessiona Acifluorfen Carfentrazone Flumioxazin Fluthiacet Lactofen Pyraflufen Saflufenacil

---------------------------------------------------------------%----------------------------------------------------

14CRI-C 100c (100)d 84 (99) 100 (100) 71f 93 (95) 94f 98 (100)

14MIS-H 55 (97) 67 (81) 66 (75) 18 42 (73) 37 79 (100)

15CLA-A 38 (64) 48 (45) 55 (44) 13 23 (66) 29 50 (98)

15CRI-A 68 (94) 54 (55) 74 (90) 39 49 (72) 53 73 (100)

15CRI-C 91 (100) 74 (87) 100 (100) 48 67 (92) 73 92 (100)

15CRI-D 72 (98) 60 (66) 91 (98) 19 44 (75) 60 93 (100)

15GRE-A 31 (78) 66 (72) 48 (81) 21 21 (29) 35 38 (99)

15IND-A 70 (94) 63 (74) 87 (90) 44 70 (79) 64 78 (100)

15LAW-C 73 (93) 58 (84) 81 (98) 33 49 (80) 57 91 (99)

15MIS-D 83 (99) 63 (80) 80 (60) 31 45 (77) 34 89 (100)

15MIS-E 73 (86) 69 (66) 79 (91) 30 54 (78) 54 95 (100)

15MIS-F 60 (93) 67 (67) 68 (89) 21 39 (69) 43 84 (100)

179

179

Table 3 (Cont.)

Mortalityb

Accessiona Acifluorfen Carfentrazone Flumioxazin Fluthiacet Lactofen Pyraflufen Saflufenacil

---------------------------------------------------------------%----------------------------------------------------

SSf 100 (100) 100 (100) 100 (100) 100 100 (100) 100 100 (100)

LSD0.05g 14 (15) 19 (19) 15 (19) 23 16 (23) 22 12

a Accessions were collected between 2014 and 2015 in Arkansas, USA. Accessions were confirmed resistant to foliar-

applied fomesafen (263 g ha-1) except for the susceptible standard (SS).

b Mortality ratings from acifluorfen, carfentrazone, flumioxazin, lactofen, and saflufenacil treatments were different across

runs, thus data were analyzed separately. Carfentrazone (280 g ha-1), pyraflufen-ethyl (3.64 g ha-1), and flumioxazin (70.6

g ha-1) treatments included 0.25% v/v non-ionic surfactant (NIS). Acifluorfen at 560 g ha-1 included 0.125% v/v NIS.

Lactofen (224 g ha-1) was sprayed with 0.5% v/v crop oil concentrate. Saflufenacil (24.6 g ha-1) was applied with 1%

methylated seed oil and 1% ammonium sulfate.

c Values obtained from the first run of experiment. Herbicides were applied to 10-cm tall seedlings.

d Values obtained from the second run of experiment. Herbicides were applied to 5- to 8-cm tall seedlings.

eAveraged mortality across two runs. Mortality ratings across runs were similar in pyraflufen-ethyl and fluthiacet-methyl treatments,

thus data were combined.

f SS = sensitive standard population

g Fisher’s protected LSD to compare accessions within each herbicide

180

Table 4. Herbicide resistance profile of PPO-resistant Palmer amaranth accessions to other

foliar-applied non-PPO herbicides.

Accession

Mortalitya

Fomesafen Dicamba Glyphosate Glufosinate Trifloxysulfuron

--------------------------------------------%--------------------------------------------

14CLA-D 62 97 91 100 85

14CRI-C 82 86 6 100 1

14CRI-G 77 93 85 100 2

14JAC-B 88 97 100 100 61

14LEE-G 86 85 95 100 49

14LEE-J 85 100 0 100 46

14MIS-A 85 97 24 100 44

14MIS-E 69 91 52 100 64

14MIS-G 85 100 89 100 38

14MIS-H 47 98 0 100 67

14PHI-B 83 100 52 100 42

15CLA-A 62 86 14 100 17

15CRI-A 40 87 5 95 7

15CRI-B 77 87 61 91 3

15CRI-C 55 93 5 100 6

15CRI-D 44 76 11 100 4

15GRE-A 23 79 93 99 10

15IND-A 43 83 14 100 22

15LAW-B 88 85 46 96 2

15LAW-C 46 84 13 97 13

15MIS-B 80 87 14 100 1

15MIS-C 64 88 9 99 5

15MIS-D 58 81 14 100 1

15MIS-E 65 84 14 100 8

15MIS-F 56 87 7 100 5

15PHI-A 74 83 2 100 11

15PRA-A 79 79 18 99 11

aFomesafen (263 g ai ha-1), dicamba (280 g ae ha-1), glufosinate (549 g ai ha-1), glyphosate (870

g ae ha-1), and trifloxysulfuron (7.84 g ha-1) were applied to 7.6-cm tall seedlings.

181

Table 5. GR50 values of PPO-resistant Palmer amaranth accessions in Arkansas.

Gly210 mutation carrier Accession GR50a R/S

g ai ha-1

Yes 14MIS-H 232

(148-315)b

15

Yes 15MIS-C 116

(80-153)

8

Yes 15MIS-E 141

(94-188)

9

No 14CRI-C 70

(43-97)

4

No 14CRI-G 153

(112-195)

10

No 15CRI-B 125

(85-164)

8

No 15PHI-A 51

(32-70)

3

No SS 15

(10-21)

1

aGR50: dose of herbicide required to cause 50% biomass reduction bValues in parenthesis indicate 95% confidence intervals.

182

Figure 1. Variability in response to foliar-applied fomesafen (263 g ha-1) among Palmer

amaranth accessions collected between 2008 and 2015.Box plot shows median values (horizontal

line inside the box), mean values (marked X), first and third quartile values (box-outlines),

minimum and maximum values (whiskers), and outlier values (open circles).

183

Figure 2. Frequency of fomesafen-resistant plants in Palmer amaranth accessions collected in

2015 from Arkansas.Fomesafen at 263 g ha-1 was applied with 0.5% non-ionic surfactant to 7.6-

cm tall seedlings. Survivors are categorized based on visible injury.

184

Figure 3. Hierarchal clustering of 2014 and 2015 Palmer amaranth accessions treated with foliar-

applied fomesafen at 263 g ha-1.The herbicide was applied with 0.5% non-ionic surfactant to 7.6-

cm tall seedlings. Cluster 1 = most sensitive to fomesafen (50 accessions), Cluster 2 = resistant

to fomesafen with majority of survivors incurred >60% injury (19 accessions), Cluster 3 =

resistant to fomesafen, majority of survivors sustained 11-30% injury (2 accessions), Cluster 4 =

resistant to fomesafen, majority of survivors incurred 0-10% injury (3 accessions).

185

Figure 4. (A) Cluster analysis of mortality levels in Palmer amaranth accessions collected in

2014 and 2015 treated with 870 g ha-1 glyphosate. Cluster 1 (n = 13 accessions; resistant to

glyphosate with majority of survivors incurred 31-60% injury), cluster 2 (n = 23 accessions;

resistant to glyphosate with majority of survivors incurred <11% injury), cluster 3 (n = 23

accessions; least sensitive to glyphosate), and cluster 4 (n = 14 accessions; resistant to glyphosate

with majority of its survivors incurred 61-89% injury) are depicted in box-plot. Glyphosate was

applied to 7.6-cm tall seedlings. (B) Variability in response to dicamba (280 g ae ha-1),

glufosinate (549 g ha-1) and trifloxysulfuron (7.84 g ha-1) among Palmer amaranth accessions

collected in 2014 and 2015 from Arkansas, USA. Box plot shows median values (horizontal line

inside the box), mean values (marked with X), first and third quartile values (box-outlines),

minimum and maximum values (whiskers), and outlier values (open circles).

186

Figure 5. Herbicide resistance profiles of Palmer amaranth populations from Arkansas sampled

in 2014 and 2015.

187

Figure 13. Counties with confirmed PPO-resistant Palmer amaranth in Arkansas.Counties that

are shaded had at least one field-population with PPO-resistant Palmer amaranth biotypes

carrying the Gly210 deletion in the PPO gene.

188

Figure 7. Prevalence of PPO Gly210 mutation in PPO-resistant Palmer amaranth from Arkansas.

(A) Pie-chart displaying the percentage of PPO-resistant accessions that contained and lacked the

PPO Gly210 mutation. Black = all fomesafen survivors in these accessions contained the Gly210

mutation. White = all survivors in these accessions lacked the Gly210 mutation. Gray = survivors

in these accessions contained mixture of carriers and non-carriers of Gly210 mutation. (B)

Proportion of Gly210 mutation carriers in each PPO-resistant accession.

189

CONCLUSIONS

Majority of the Palmer amaranth accessions from Arkansas were sensitive to glufosinate

but resistant to trifloxysulfuron and glyphosate. Increased expression of GST and CYPA219

genes were associated with differential tolerance to glufosinate. The elevated expression of these

detoxification-related genes can get fixed in population with sustained selection pressure, leading

to evolution of resistance. Resistance to ALS inhibitors in a Palmer amaranth population from

Arkansas is attributed to ALS Ser653Asn mutation and elevated levels of CYP81B and GSTF10

genes. Induced expression of these non-target genes endow resistance to various ALS herbicide

classes.

Palmer amaranth in Arkansas had evolved resistance to PPO-inhibiting herbicides. PPO-

resistant Palmer amaranth biotype was first detected in a population collected in 2011. (4) Since

then, the number of PPO-resistant populations increased in at least 13 counties in Arkansas in

2015. Fomesafen-resistant populations were generally resistant to trifloxysulfuron and

glyphosate and were also cross-resistant to other PPO-inhibiting herbicides such as

carfentrazone, flumioxazin, and lactofen. The PPO Gly210 deletion is the prevalent (55%)

resistance mechanism among PPO-resistant Palmer amaranth populations from Arkansas. Plants

carrying PPO Gly210 deletion or Arg128Gly mutations had similar level of resistance to

fomesafen. Palmer amaranth has multiple genetic weapons, within or outside of the target site,

which it can use to counteract the lethal effects of herbicides. Resistance to multiple modes of

action, coupled with target and non-target-site mechanisms, severely limits herbicide option for

Palmer amaranth control. With Palmer amaranth being an obligate crossing species, plants in a

population can accumulate multiple resistance mechanisms which would likely drive resistance

evolution faster and is therefore detrimental to chemical weed management.

non-target-site based tolerance to herbicides in

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